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.     

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。     

Proteomic analysis in clear cell renal cell carcinoma: identification of differentially expressed protein by 2-D DIGE

Renal cell carcinoma (RCC), the most common neoplasm affecting the adult kidney, is characterised by heterogeneity of histological subtypes, drug resistance, and absence of molecular markers. Two-dimensional difference gel electrophoresis (2-D DIGE) technology in combination with mass spectrometry (MS) was applied to detect differentially expressed proteins in 20 pairs of RCC tissues and matched adjacent normal kidney cortex (ANK), in order to search for RCC markers. After gel analysis by DeCyder 6.5 and EDA software, differentially expressed protein spots were excised from Deep Purple stained preparative 2DE gel. A total of 100 proteins were identified by MS out of 2500 spots, 23 and 77 of these were, respectively, over- and down-expressed in RCC. The Principal Component Analysis applied to gels and protein spots exactly separated the two sample classes in two groups: RCC and ANK. Moreover, some spots, including ANXA2, PPIA, FABP7 and LEG1, resulted highly differential. The DIGE data were also confirmed by immunoblotting analysis for these proteins. In conclusion, we suggest that applying 2-D DIGE to RCC may provide the basis for a better molecular characterization and for the discovery of candidate biomarkers.     

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.     

Toward a comprehensive quantitative proteome database: protein expression map of lymphoid neoplasms by 2-D DIGE and MS

Using 2-D DIGE, we constructed a quantitative 2-D database including 309 proteins corresponding to 389 protein spots across 42 lymphoid neoplasm cell lines. The proteins separated by 2-D PAGE were identified by MS and assigned to the expression data obtained by 2-D DIGE. The cell lines were categorized into four groups: those from Hodgkin's lymphoma (HL) (4 cell lines), B cell malignancies (19 cell lines), T cell malignancies (16 cell lines), and natural killer (NK) cell malignancies (3 cell lines). We characterized the proteins in the database by classifying them according to their expression level. We found 28 proteins with more than a 2-fold difference between the cell line groups. We also noted the proteins that allowed multidimensional separation to be achieved (1) between HL cells and other cells, (2) between the cells derived from B cells, T cells and NK cells, and (3) between HL cells and anaplastic large cell lymphoma cells. Decision tree classification identified five proteins that could be used to classify the 42 cell lines according to differentiation. These results suggest that the quantitative 2-D database using 2-D DIGE will be a useful resource for studying the mechanisms underlying the differentiation phenotypes of lymphoid neoplasms.     

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 the HUPO Brain Proteome reference samples using 2-D DIGE and 2-D LC-MS/MS

Within the Human Proteome Organization (HUPO) Brain Proteome Project, a pilot study was launched with reference samples shipped to nine international laboratories (see Hamacher et al., this Special Issue) to evaluate different proteome approaches in neuroscience and to build up a first version of a brain protein database. One part of the study addresses quantitative proteome alterations between three developmental stages (embryonic day 16; postnatal day 7; 8 weeks) of mouse brains. Five brains per stage were differentially analyzed by 2-D DIGE using internal standardization and overlapping pH gradients (pH 4-7 and 6-9). In total, 214 protein spots showing stage-dependent intensity alterations (> two-fold) were detected, 56 of which were identified. Several of them, e.g. members of the dihydropyrimidinase family, are known to be associated with brain development. To feed the HUPO BPP brain protein database, a robust 2-D LC-MS/MS method was applied to murine postnatal day 7 and human post-mortem brain samples. Using MASCOT and the IPI database, 350 human and 481 mouse proteins could be identified by at least two different peptides. The data are accessible through the PRIDE database (http://www.ebi.ac.uk/pride/).     

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.     

Characterization of proteins in human pancreatic cancer serum using differential gel electrophoresis and tandem mass spectrometry

The purpose of this study was to develop techniques for identifying cancer biomarkers in human serum using differential in-gel electrophoresis (DIGE), and characterizing the protein biomarkers using tandem mass spectrometry (MS/MS). A major problem in profiling protein expression by DIGE comes from the presence of high concentrations of a small number of proteins. Therefore, serum samples were first chromatographed using an immunoaffinity HPLC column (Agilent Technologies), to selectively remove albumin, immunoglobulins, transferrin, haptoglobin, and antitrypsin. Serum samples from three individuals with pancreatic cancer and three individuals without cancer were compared. Serum samples were processed using the immunoaffinity column. Differential protein analysis was performed using DIGE. A total of 56 protein spot-features were found to be significantly increased and 43 significantly decreased in cancer serum samples. These spot features were excised, trypsin digested, and analyzed by MALDI/TOF/TOF (4700 Proteomics Analyzer, Applied Biosystems). We identified 24 unique proteins that were increased and 17 unique proteins that were decreased in cancer serum samples. Western blot analysis confirmed increased levels of several of these proteins in the pancreatic cancer serum samples. In an independent series of serum samples from 20 patients with pancreatic cancer and 14 controls, increased levels of apolipoprotein E, alpha-1-antichymotrypsin, and inter-alpha-trypsin inhibitor were found to be associated with pancreatic cancer. These results suggest that affinity column enrichment and 2-D DIGE can be used to identify numerous proteins differentially expressed in serum from individuals with pancreatic cancer.     

Proteomic analysis of p38alpha mitogen-activated protein kinase-regulated changes in membrane fractions of RAS-transformed fibroblasts

Oncogenic Ras signaling has been long known to play an important role in tumorigenesis and human cancer. In this report, we have used the sensitive 2-D-DIGE coupled to MS for the identification of proteins differentially expressed at the cell membrane level between oncogenic H-RasV12-transformed wild-type and p38alpha-deficient mouse embryo fibroblasts (MEFs). Following trifluoroethanol solubilization, 76 proteins were found to be differentially regulated. After PMF, 63 spots containing 42 different proteins were unequivocally identified by MALDI-TOF MS coupled with database interrogation. As expected, many of them were membrane proteins. Six proteins were selected for further validation studies based on their potential functional link with malignant transformation and signal transduction. These were prohibitin (PHB), protein disulfide isomerase 3 (PDIA3), focal adhesion kinase 2 (FAK2), c-GMP dependent protein kinase 2 (KGP2), NADH-ubiquinone oxidoreductase 30 kDa subunit (NUGM) and translationally controlled tumor protein (TCTP). All these proteins were up-regulated in the membranes of H-RasV12-transformed p38alpha-/-cells, except for prohibitin, which was down-regulated. An excellent correlation was found between DIGE results and Western blot studies, indicating the reliability of the 2-D-DIGE analysis. The available evidence about the putative function of the identified proteins supports the emerging role of p38alpha as a negative regulator of tumorigenesis. Further studies are in progress to elucidate the implications of these findings in the regulation of H-Ras-induced transformation by p38alpha signaling.     

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.     

Evaluation of the combination of bead technology with SELDI-TOF-MS and 2-D DIGE for detection of plasma proteins

Today biomarker discovery is one of the most active aspects of proteomic investigations. However, the wide dynamic range of plasma proteins makes the analysis very challenging because high abundance proteins tend to mask those of lower abundance. Using a large bead-based library of combinatorial peptide ligands (Equalizer beads or ProteoMiner), the dynamic range of the protein concentration is compressed, the high abundance proteins present in the sample are reduced and the low abundance proteins are enriched, while retaining representatives of all proteins within the sample. In the present study, the combination of beads with surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and two-dimensional differential gel electrophoresis (2-D DIGE) technology were evaluated considering efficiency, reproducibility, sensitivity, and compatibility. The bead technology is easily compatible with both SELDI-TOF-MS and 2-D DIGE and the samples can be analyzed directly without any processing of the sample. The use of the beads prior SELDI-TOF-MS and 2-D DIGE enabled detection of many new protein spots/peaks and increased resolution and improved intensity of low abundance proteins in a reproducible fashion compared with the depletion technique. Several proteins have been identified by the combination of beads, 2-D DIGE and MS for example different kinds of complement factors and cytoskeletal proteins. Our data suggest that integration of the bead technology with our current proteomic technologies will enhance the possibility to deliver new peptide/protein biomarker candidates in our projects.     

Development and plasticity-related changes in protein expression patterns in cat visual cortex: a fluorescent two-dimensional difference gel electrophoresis approach

During early postnatal brain development, changes in visual input can lead to specific alteration of function and connectivity in mammalian visual cortex. In cat, this so-called critical period exhibits maximal sensory-driven adaptations around postnatal day 30 (P30), and ceases toward adulthood. We examined the molecular framework that directs age- and experience-dependent plasticity in cat visual cortex, by comparing protein expression profiles at eye opening (postnatal day 10 (P10), when experience-dependent plasticity starts), the peak of the critical period (P30), and in adulthood. Using 2-D DIGE, we performed comparisons of P10-P30 and P30-adult brain protein samples. Sixty protein spots showed statistically significant intensity changes in at least one comparison. Fifty-one spots were identified using quadrupole-TOF MS/MS or LC-MS/MS, containing 37 different proteins. The progressive increase or decrease in protein expression levels could be correlated to age-dependent postnatal brain development. Four spots containing transferrin, 14-3-3 alpha/beta and cypin, showed maximal protein expression levels at P30, thereby showing a positive correlation to critical period plasticity. Western analysis indeed revealed a clear effect of visual deprivation on cypin expression in cat visual cortex. Our results therefore demonstrate the power of 2-D DIGE as a tool toward understanding the molecular basis of nervous system development and plasticity.     

2D differential in-gel electrophoresis for the identification of esophageal scans cell cancer-specific protein markers

The reproducibility of conventional two-dimensional (2D) gel electrophoresis can be improved using differential in-gel electrophoresis (DIGE), a new emerging technology for proteomic analysis. In DIGE, two pools of proteins are labeled with 1-(5-carboxypentyl)-1'-propylindocarbocyanine halide (Cy3) N-hydroxy-succinimidyl ester and 1-(5-carboxypentyl)-1'-methylindodi-carbocyanine halide (Cy5) N-hydroxysuccinimidyl ester fluorescent dyes, respectively. The labeled proteins are mixed and separated in the same 2D gel. 2D DIGE was applied to quantify the differences in protein expression between laser capture microdissection-procured esophageal carcinoma cells and normal epithelial cells and to define cancer-specific and normal-specific protein markers. Analysis of the 2D images from protein lysates of approximately 250,000 cancer cells and normal cells identified 1038 protein spots in cancer cell lysates and 1088 protein spots in normal cell lysates. Of the detected proteins, 58 spots were up-regulated by >3-fold and 107 were down-regulated by >3-fold in cancer cells. In addition to previously identified down-regulated protein annexin I, tumor rejection antigen (gp96) was found up-regulated in esophageal squamous cell cancer. Global quantification of protein expression between laser capture-microdissected patient-matched cancer cells and normal cells using 2D DIGE in combination with mass spectrometry is a powerful tool for the molecular characterization of cancer progression and identification of cancer-specific protein markers.     

Proteomic analysis of lymph

This report provides the first proteomic analysis of normal ovine lymph. By establishing the fact that lymph is more than an ultrafiltrate of blood plasma, it documents that the lymph proteome contains an array of proteins that differentiates it from plasma. The protein chip technology, surface-enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS), two-dimensional gel electrophoresis (2-D PAGE) and MS, were employed to examine the protein expression profiles of ovine lymph. Using a weak cation exchange chip surface to assay lymph and plasma samples by SELDI-TOF-MS showed that the analysis of peak maps from lymph contained three protein peaks that were found only in lymph, while analysis of peak maps from plasma samples showed that five protein peaks were found only in plasma. Lymph and plasma samples showed eight peaks that were common to both. There were also more ions present in plasma than in lymph, which is consistent with the 2-D PAGE analysis. MS analysis of a large number of protein spots from 2-D PAGE gels of lymph produced MS/MS sequences for 18 proteins that were identified by searching against a comprehensive protein sequence database. As in plasma, large protein spots of albumin dominated the protein pattern in lymph. Other major proteins identified in 2-D PAGE gels of lymph included, fibrinogen alpha- and beta-chains, immunoglobulin G (IgG) heavy chain, serotransferrin precursor, lactoferrin, and apolipoprotein A-1. Two proteins that were identified and were differentially expressed in lymph were glial fibrillary astrocyte acidic protein and neutrophil cytosol factor-1. By bringing the technologies of proteomics to bear on the analysis of lymph, it is possible to detect proteins in lymph that are quantitatively and qualitatively differentially expressed from those of plasma.     

Proteomic analysis of papaya (Carica papaya L.) displaying typical sticky disease symptoms

Papaya (Carica papaya L.) hosts the only described laticifer-infecting virus (Papaya meleira virus, PMeV), which is the causal agent of papaya sticky disease. To understand the systemic effects of PMeV in papaya, we conducted a comprehensive proteomic analysis of leaf samples from healthy and diseased plants grown under field conditions. First, a reference 2-DE map was established for proteins from healthy samples. A total of 486 reproducible spots were identified, and MALDI-TOF-MS/MS data identified 275 proteins accounting for 159 distinct proteins from 231 spots that were annotated. Second, the differential expression of proteins from healthy and diseased leaves was determined through parallel experiments, using 2-DE and DIGE followed by MALDI-TOF-MS/MS and LC-IonTrap-MS/MS, respectively. Conventional 2-DE analysis revealed 75 differentially expressed proteins. Of those, 48 proteins were identified, with 26 being upregulated (U) and 22 downregulated (D). In general, metabolism-related proteins were downregulated, and stress-responsive proteins were upregulated. This expression pattern was corroborated by the results of the DIGE analysis, which identified 79 differentially expressed proteins, with 23 identified (17 U and 6 D). Calreticulin and the proteasome subunits 20S and RPT5a were shown to be upregulated during infection by both 2-DE and DIGE analyses. These data may help shed light on plant responses against stresses and viral infections.     

Quantitative profiling of drug-associated proteomic alterations by combined 2-nitrobenzenesulfenyl chloride (NBS) isotope labeling and 2DE/MS identification

The identification of drug-responsive biomarkers in complex protein mixtures is an important goal of quantitative proteomics. Here, we describe a novel approach for identifying such drug-induced protein alterations, which combines 2-nitrobenzenesulfenyl chloride (NBS) tryptophan labeling with two-dimensional gel electrophoresis (2DE)/mass spectrometry (MS). Lysates from drug-treated and control samples are labeled with light or heavy NBS moiety and separated on a common 2DE gel, and protein alterations are identified by MS through the differential intensity of paired NBS peptide peaks. Using NBS/2DE/MS, we profiled the proteomic alterations induced by tamoxifen (TAM) in the estrogen receptor (ER) positive MCF-7 breast cancer cell line. Of 88 protein spots that significantly changed upon TAM treatment, 44 spots representing 23 distinct protein species were successfully identified with NBS-paired peptides. Of these 23 TAM-altered proteins, 16 (70%) have not been previously associated with TAM or ER activity. We found the NBS labeling procedure to be both technically and biologically reproducible, and the NBS/2DE/MS alterations exhibited good concordance with conventional 2DE differential protein quantitation, with discrepancies largely due to the comigration of distinct proteins in the regular 2DE gels. To validate the NBS/2DE/MS results, we used immunoblotting to confirm GRP78, CK19, and PA2G4 as bona fide TAM-regulated proteins. Furthermore, we demonstrate that PA2G4 expression can serve as a novel prognostic factor for disease-free survival in two independent breast cancer patient cohorts. To our knowledge, this is the first report describing the proteomic changes in breast cancer cells induced by TAM, the most commonly used selective estrogen receptor modulator (SERM). Our results indicate that NBS/2DE/MS may represent a more reliable approach for cellular protein quantitation than conventional 2DE approaches.     

Poly(ADP-Ribose) Polymerase 1 (PARP1) Overexpression in Human Breast Cancer Stem Cells and Resistance to Olaparib

Background

Breast cancer stem cells (BCSCs) have been recognized as playing a major role in various aspects of breast cancer biology. To identify specific biomarkers of BCSCs, we have performed comparative proteomics of BCSC-enriched and mature cancer cell populations from the human breast cancer cell line (BCL), BrCA-MZ-01.

Methods

ALDEFLUOR assay was used to sort BCSC-enriched (ALDH+) and mature cancer (ALDH−) cell populations. Total proteins were extracted from both fractions and subjected to 2-Dimensional Difference In-Gel Electrophoresis (2-D DIGE). Differentially-expressed spots were excised and proteins were gel-extracted, digested and identified using MALDI-TOF MS.

Results

2-D DIGE identified poly(ADP-ribose) polymerase 1 (PARP1) as overexpressed in ALDH+ cells from BrCA-MZ-01. This observation was confirmed by western blot and extended to four additional human BCLs. ALDH+ cells from BRCA1-mutated HCC1937, which had the highest level of PARP1 overexpression, displayed resistance to olaparib, a specific PARP1 inhibitor.

Conclusion

An unbiased proteomic approach identified PARP1 as upregulated in ALDH+, BCSC-enriched cells from various human BCLs, which may contribute to clinical resistance to PARP inhibitors.