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Two-dimensional Difference Gel Electrophoresis (two-dimensional + difference_gel_electrophoresis)
Selected AbstractsEvaluation of saturation labelling two-dimensional difference gel electrophoresis fluorescent dyesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 7 2003Joanne Shaw Abstract Two-dimensional difference gel electrophoresis (2-D DIGE) enables an increased confidence in detection of protein differences. However, due to the nature of the minimal labelling where only approximately 5% of a given protein is labelled, spots cannot be directly excised for mass spectrometry (MS) analysis and detection sensitivity could be further enhanced. Amersham Biosciences have developed a second set of CyDyeÔ DIGE CyÔ3 and Cy5 dyes, which aim to overcome these limitations through saturation-labelling of cysteine residues. The dyes were evaluated in relation to their sensitivity and dynamic range, their useability as multiplexing reagents and the possibility of direct spot picking from saturation-labelled gels for MS analysis. The saturation-labelling dyes were superior in sensitivity to their minimal-labelling counterparts, silver stain and Sypro Ruby, however, the resulting 2-D spot pattern was significantly altered from that of unlabelled or minimal-labelled protein. The dyes were found to be useful as multiplexing reagents although preferential labelling of proteins with one dye over another was observed but was controlled for through experimental design. Protein identities were successfully obtained from material directly excised from saturation-labelled gels eliminating the need for post-stained preparative gels. [source] Tetranectin and apolipoprotein A-I in cerebrospinal fluid as potential biomarkers for Parkinson's diseaseACTA NEUROLOGICA SCANDINAVICA, Issue 5 2010E.-S. Wang Wang E-S, Sun Y, Guo J-G, Gao X, Hu J-W, Zhou L, Hu J, Jiang C-C. Tetranectin and apolipoprotein A-I in cerebrospinal fluid as potential biomarkers for Parkinson's disease. Acta Neurol Scand: 2010: 122: 350,359. © 2010 The Authors Journal compilation © 2010 Blackwell Munksgaard. Objective,,, The application of biomarkers may potentially improve the efficiency of the diagnosis for Parkinson's disease (PD). However, no reliable biomarker has been identified to date. This study is aimed to identify proteins that might serve as potential biomarkers for PD diagnosis or pathogenesis. Materials and methods,,, Two-dimensional difference gel electrophoresis (2D DIGE) technique, in combination with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), was used to determine the differentially expressed cerebrospinal fluid (CSF) proteins in PD patients (n = 3) compared with normal controls (n = 3). Selected proteins were further confirmed by Western blotting analysis in the CSF of PD patients (n = 8), Alzheimer's disease (AD) patients (n = 6) and normal control subjects (n = 7). Results,,, Eight proteins were identified after MS and protein database interrogation. In the CSF of PD patients, the expression levels of one isoform of apolipoprotein A-I (apoA-I), tetranectin, myosin phosphatase target subunit 1 (MYPT1), and two unknown proteins were down-regulated, whereas the expression levels of another apoA-I isoform, proapolipoprotein, and lipoprotein were up-regulated. Western blotting indicates that the expression of tetranectin was reduced in the CSF from PD patients and elevated in AD, while the expression of apoA-I was changed only in the CSF from PD patients. Conclusion,,, Our preliminary results suggest that tetranectin and apoA-I may serve as potential biomarkers for PD, though further validation is needed. [source] Proteomic profiling reveals the prognostic value of adenomatous polyposis coli,end-binding protein 1 in hepatocellular carcinoma,HEPATOLOGY, Issue 6 2008Tatsuya Orimo Histological differentiation is a major pathological parameter associated with poor prognosis in patients with hepatocellular carcinoma (HCC) and the molecular signature underlying HCC differentiation may involve key proteins potentially affecting the malignant characters of HCC. To develop prognostic biomarkers for HCC, we examined the global protein expression profiles of 45 surgically resected tissues, including 27 HCCs with different degree of histological differentiation, 11 adjacent nontumor tissues, and seven normal liver tissues. Unsupervised classification grouped the 45 samples according to their histological classification based on the protein expression profiles created by laser microdissection and two-dimensional difference gel electrophoresis (2D-DIGE). Statistical analysis and mass spectrometry identified 26 proteins with differential expression, of which 14 were functionally linked to c-Myc, AP-1, HIF1A, hepatocyte nuclear factor 4 alpha, or the Ras superfamily (RhoA, CDC42, and Rac1). Among the proteins identified, we focused on APC-binding protein EB1 (EB1) because it was dominantly expressed in poorly differentiated HCCs, which generally correlate with the poor prognosis in patients with HCC. In addition, EB1 is controlled by c-Myc, RhoA, and CDC42, which have all been linked to HCC malignancy. Immunohistochemistry in a further 145 HCC cases revealed that EB1 significantly correlated with the degree of histological differentiation (P < 0.001), and univariate and multivariate analyses indicated that EB1 is an independent prognostic factor for recurrence (hazard ratio, 2.740; 95% confidence interval, 1.771,4.239; P < 0.001) and survival (hazard ratio, 2.256; 95% confidence interval, 1.337,3.807; P = 0.002) of patients with HCC after curative surgery. Conclusion: Proteomic profiling revealed the molecular signature behind the progression of HCC, and the prognostic value of EB1 in HCC. (HEPATOLOGY 2008;48:1851-1863.) [source] Transglutaminase 3 as a prognostic biomarker in esophageal cancer revealed by proteomicsINTERNATIONAL JOURNAL OF CANCER, Issue 9 2009Norihisa Uemura Abstract To develop a prognostic biomarker for esophageal squamous cell carcinoma (ESCC), we examined the proteomic profile of ESCC using two-dimensional difference gel electrophoresis (2D-DIGE), and identified proteins associated with prognosis by mass spectrometry. The prognostic performance of the identified proteins was examined by immunohistochemistry in additional cases. We identified 22 protein spots whose intensity was statistically different between ESCC cases with good (N = 9; survived more than 5 years without evidence of recurrence) and poor (N = 24; died within 2 years postsurgery) prognosis, within the patient group that had two or more lymph node metastases. Mass spectrometric protein identification resulted in 18 distinct gene products from the 22 protein spots. Transglutaminase 3 (TGM3) was inversely correlated with shorter patient survival. The prognostic performance of TGM3 was further examined by immunohistochemistry in 76 ESCC cases. The 5-year disease-specific survival rate was 64.5% and 32.1% for patients with TGM3-positive and TGM3-negative tumors, respectively (p = 0.0033). Univariate and multivariate analyses revealed that TGM3 expression was an independent prognostic factor among the clinicopathologic variables examined. It is noteworthy that the prognostic value of TGM3 was shown to be higher than those of the lymph node metastasis, intramural metastasis and vascular invasion status. These results establish TGM3 as a novel prognostic biomarker for ESCC for the first time. Examination of TGM3 expression may provide novel therapeutic strategies to prevent recurrence of ESCC. © 2008 Wiley-Liss, Inc. [source] Plasma proteomics of lung cancer by a linkage of multi-dimensional liquid chromatography and two-dimensional difference gel electrophoresisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 13 2006Tetsuya Okano Abstract To investigate aberrant plasma proteins in lung cancer, we compared the proteomic profiles of serum from five lung cancer patients and from four healthy volunteers. Immuno-affinity chromatography was used to deplete highly abundant plasma proteins, and the resulting plasma samples were separated into eight fractions by anion-exchange chromatography. Quantitative protein profiles of the fractionated samples were generated by two-dimensional difference gel electrophoresis, in which the experimental samples and the internal control samples were labeled with different dyes and co-separated by two-dimensional polyacrylamide gel electrophoresis. This approach succeeded in resolving 3890 protein spots. For 364 of the protein spots, the expression level in lung cancer was more than twofold different from that in the healthy volunteers. These differences were statistically significant (Student's t -test, p -value less than 0.05). Mass spectrometric protein identification revealed that the 364 protein spots corresponded to 58 gene products, including the classical plasma proteins and the tissue-leakage proteins catalase, clusterin, ficolin, gelsolin, lumican, tetranectin, triosephosphate isomerase and vitronectin. The combination of multi-dimensional liquid chromatography and two-dimensional difference gel electrophoresis provides a valuable tool for serum proteomics in lung cancer. [source] Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 11 2005Hong-Lin Chan Abstract Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N -hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes. Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response. [source] Proteomic study of human hepatocellular carcinoma using two-dimensional difference gel electrophoresis with saturation cysteine dyePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2005Kazuyasu Fujii Abstract To identify the proteomic alterations associated with carcinogenesis of hepatocellular carcinoma (HCC), we compared the protein expression profiles of nine HCC cell lines with those of primary cultured hepatocytes established from five individuals. A differential proteomic study was performed by two-dimensional difference gel electrophoresis, in which protein samples are labeled with different fluorescent dyes and separated according to their isoelectric point and molecular weight. To label the protein samples, we used a newly developed and highly sensitive fluorescent dye, which reacts with all reduced cysteine residues of proteins. Principal component analysis based on the intensity of 1238,protein spots indicated that the HCC cells and the normal hepatocytes had distinct proteomic profiles. The Wilcoxon test was used to determine the protein spots whose intensity was differentially regulated in the HCC cells compared with the normal hepatocytes, and mass spectrometric analysis was used to identify the proteins corresponding to the spots. The proteins identified are involved in cell cycle regulation, binding to a tumor-suppressor gene product, fatty acid binding, and regulation of translation. Western blotting with specific antibodies revealed the overexpression of PCNA, EB1 and E-FABP in HCC tissues compared with noncancerous tissues. Aberrant regulation of EB1 and E-FABP has not previously been implicated in the development of HCC. [source] Proteins specifically hyperexpressed in a coeliac disease patient with aberrant T cellsCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 3 2007V. De Re Summary An aberrant T cell population is the basis for diagnosis of refractory coeliac disease and determines the risk of enteropathy-associated T cell lymphoma. This disease is serious with a poor survival. Pathogenetic mechanisms sustaining aberrant T cell proliferation remain unknown. Recently, alemtuzumab has been proposed as a promising new approach to treat these patients. Only few single cases have been tested at present; nevertheless, in all the cases a clinical improvement was observed. However, whether intraepithelial lymphocytes have been targeted effectively by alemtuzumab is still debated. This study reports, using two-dimensional difference gel electrophoresis (2D DIGE), hyperexpressed proteins associated specifically with aberrant T cells found in a patient with coeliac disease by comparison of the protein expression of this sample with that of patients with coeliac disease and polyclonal T cells or with control subjects. The data demonstrated a significantly higher expression of IgM, apolipoprotein C-III and Charcot,Leyden crystal proteins in a duodenal biopsy specimen of the patient with clonal T cells compared with that of other patients. These preliminary results allow hypothesizing different clinical effects of alemtuzumab in patients with coeliac disease and aberrant T cell proliferation, because as well as the probable effect on T cells, alemtuzumab could exert its effect by acting on inflammatory associated CD52+ IgM+ B cells and eosinophil cells, known to produce IgM and Charcot,Leyden crystal proteins, that we demonstrated to be altered in this patient. The results also emphasize the possible association of apolipoprotein with aberrant T cell proliferation. [source] | ||||||||||