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Quantitative Proteomic Analysis (quantitative + proteomic_analysis)
Selected AbstractsQuantitative proteomic analysis to discover potential diagnostic markers and therapeutic targets in human renal cell carcinomaPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 15 2008Noboru Okamura Abstract Renal cell carcinoma (RCC) is relatively resistant to chemotherapy and radiotherapy. Recent advances in drug development are providing novel agents for the treatment of RCC, but the effects are still minimal. In addition, there is an urgent need to identify diagnostic markers for RCC. In this report, to discover potential diagnostic markers and therapeutic targets, we subjected RCC samples to a quantitative proteomic analysis utilizing 2-nitrobenzenesulfenyl (NBS) reagent. Proteins were extracted from RCC and adjacent normal tissue, obtained surgically from patients, and labeled with NBS reagent containing six 12C or 13C. This was followed by trypsin digestion and the enrichment of labeled peptides. Samples were then subjected to analysis by MALDI-TOF MS. NBS-labeled peptides with a 6,Da difference were identified by MS/MS. Thirty-four proteins were upregulated in more than 60% of the patients of which some were previously known, and some were novel. The identity of a few proteins was confirmed by Western blotting and quantitative real time RT-PCR. The results suggest that NBS-based quantitative proteomic analysis is useful for discovering diagnostic markers and therapeutic targets for RCC. [source] Quantitative proteomics of intracellular Porphyromonas gingivalisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 23 2007Qiangwei Xia Abstract Whole-cell quantitative proteomic analyses were conducted to investigate the change from an extracellular to intracellular lifestyle for Porphyromonas gingivalis, a Gram-negative intracellular pathogen associated with periodontal disease. Global protein abundance data for P. gingivalis strain ATCC 33277 internalized for 18,h within human gingival epithelial cells and controls exposed to gingival cell culture medium were obtained at sufficient coverage to provide strong evidence that these changes are profound. A total of 385 proteins were overexpressed in internalized P. gingivalis relative to controls; 240 proteins were shown to be underexpressed. This represented in total about 28% of the protein encoding ORFs annotated for this organism, and slightly less than half of the proteins that were observed experimentally. Production of several proteases, including the classical virulence factors RgpA, RgpB, and Kgp, was decreased. A separate validation study was carried out in which a 16-fold dilution of the P. gingivalis proteome was compared to the undiluted sample in order to assess the quantitative false negative rate (all ratios truly alternative). Truly null (no change) abundance ratios from technical replicates were used to assess the rate of quantitative false positives over the entire proteome. A global comparison between the direction of abundance change observed and previously published bioinformatic gene pair predictions for P. gingivalis will assist with future studies of P. gingivalis gene regulation and operon prediction. [source] Separation with zwitterionic hydrophilic interaction liquid chromatography improves protein identification by matrix-assisted laser desorption/ionization-based proteomic analysisBIOMEDICAL CHROMATOGRAPHY, Issue 6 2009Atsushi Intoh Abstract Comprehensive proteomic analyses necessitate efficient separation of peptide mixtures for the subsequent identification of proteins by mass spectrometry (MS). However, digestion of proteins extracted from cells and tissues often yields complex peptide mixtures that confound direct comprehensive MS analysis. This study investigated a zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) technique for the peptide separation step, which was verified by subsequent MS analysis. Human serum albumin (HSA) was the model protein used for this analysis. HSA was digested with trypsin and resolved by ZIC-HILIC or conventional strong cation exchange (SCX) prior to MS analysis for peptide identification. Separation with ZIC-HILIC significantly improved the identification of HSA peptides over SCX chromatography. Detailed analyses of the identified peptides revealed that the ZIC-HILIC has better peptide fractionation ability. We further demonstrated that ZIC-HILIC is useful for quantitatively surveying cell surface markers specifically expressed in undifferentiated embryonic stem cells. These results suggested the value of ZIC-HILIC as a novel and efficient separation method for comprehensive and quantitative proteomic analyses. Copyright © 2009 John Wiley & Sons, Ltd. [source] Oncoproteomics of hepatocellular carcinoma: from cancer markers' discovery to functional pathwaysLIVER INTERNATIONAL, Issue 8 2007Stella Sun Abstract Hepatocellular carcinoma (HCC) is a heterogeneous cancer with no promising treatment and remains one of the most prevailing and lethal malignancies in the world. Researchers in many biological areas now routinely identify and characterize protein markers by a mass spectrometry-based proteomic approach, a method that has been commonly used to discover diagnostic biomarkers for cancer detection. The proteomic research platforms span from the classical two-dimensional polyacrylamide gel electrophoresis (2-DE) to the latest Protein Chip or array technology, which are often integrated with the MALDI (matrix-assisted laser-desorption ionization), SELDI (surface-enhanced laser desorption/ionization) or tandem mass spectrometry (MS/MS). New advances on quantitative proteomic analysis (e.g. SILAC, ICAT, and ITRAQ) and multidimensional protein identification technology (MudPIT) have greatly enhanced the capability of proteomic methods to study the expressions, modifications and functions of protein markers. The present article reviews the latest proteomic development and discovery of biomarkers in HCC that may provide insights into the underlying mechanisms of hepatocarcinogenesis and the readiness of biomarkers for clinical uses. [source] Comparative proteomics of human embryonic stem cells and embryonal carcinoma cellsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 7 2010Raghothama Chaerkady Abstract Pluripotent human embryonic stem cells (ESCs) can be differentiated in vitro into a variety of cells which hold promise for transplantation therapy. Human embryonal carcinoma cells (ECCs), stem cells of human teratocarcinomas, are considered a close but malignant counterpart to human ESCs. In this study, a comprehensive quantitative proteomic analysis of ESCs and ECCs was carried out using the iTRAQ method. Using two-dimensional LC and MS/MS analyses, we identified and quantitated ,1800 proteins. Among these are proteins associated with pluripotency and development as well as tight junction signaling and TGF, receptor pathway. Nearly ,200 proteins exhibit more than twofold difference in abundance between ESCs and ECCs. Examples of early developmental markers high in ESCs include ,-galactoside-binding lectin, undifferentiated embryonic cell transcription factor-1, DNA cytosine methyltransferase 3, isoform-B, melanoma antigen family-A4, and interferon-induced transmembrane protein-1. In contrast, CD99-antigen (CD99), growth differentiation factor-3, cellular retinoic acid binding protein-2, and developmental pluripotency associated-4 were among the highly expressed proteins in ECCs. Several proteins that were highly expressed in ECCs such as heat shock 27,kDa protein-1, mitogen-activated protein kinase kinase-1, nuclear factor of , light polypeptide gene enhancer in B-cells inhibitor like-2, and S100 calcium-binding protein-A4 have also been attributed to malignancy in other systems. Importantly, immunocytochemistry was used to validate the proteomic analyses for a subset of the proteins. In summary, this is the first large-scale quantitative proteomic study of human ESCs and ECCs, which provides critical information about the regulators of these two closely related, but developmentally distinct, stem cells. [source] iTRAQ quantitative analysis of Francisella tularensis ssp. holarctica live vaccine strain and Francisella tularensis ssp. tularensis SCHU S4 response to different temperatures and stationary phases of growthPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 10 2009Juraj Lenco Abstract Proteomics has been shown to significantly contribute to the investigation of the pathogenicity of the extremely infectious bacteria Francisella tularensis. In this study, the authors employed iTRAQ quantitative proteomic analysis in order to monitor alterations in proteomes of F. tularensis ssp. holarctica live vaccine strain and F. tularensis ssp. tularensis SCHU S4 associated with the cultivation at different temperatures or in the stationary phase. Correlated production of the identified proteins studied by the exploratory statistical analysis revealed novel candidates for virulence factors that were regulated in a similar manner to the genes encoded in the Francisella Pathogenicity Island. Moreover, the assessment of the adaptation of live vaccine strain and SCHU S4 strain to the examined stimuli uncovered differences in their physiological responses to the stationary phase of growth. [source] Quantitative proteomic analysis to discover potential diagnostic markers and therapeutic targets in human renal cell carcinomaPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 15 2008Noboru Okamura Abstract Renal cell carcinoma (RCC) is relatively resistant to chemotherapy and radiotherapy. Recent advances in drug development are providing novel agents for the treatment of RCC, but the effects are still minimal. In addition, there is an urgent need to identify diagnostic markers for RCC. In this report, to discover potential diagnostic markers and therapeutic targets, we subjected RCC samples to a quantitative proteomic analysis utilizing 2-nitrobenzenesulfenyl (NBS) reagent. Proteins were extracted from RCC and adjacent normal tissue, obtained surgically from patients, and labeled with NBS reagent containing six 12C or 13C. This was followed by trypsin digestion and the enrichment of labeled peptides. Samples were then subjected to analysis by MALDI-TOF MS. NBS-labeled peptides with a 6,Da difference were identified by MS/MS. Thirty-four proteins were upregulated in more than 60% of the patients of which some were previously known, and some were novel. The identity of a few proteins was confirmed by Western blotting and quantitative real time RT-PCR. The results suggest that NBS-based quantitative proteomic analysis is useful for discovering diagnostic markers and therapeutic targets for RCC. [source] Selective isolation of multiple positively charged peptides for 2-DE-free quantitative proteomicsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 16 2006Aniel Sánchez Abstract A method for quantitative proteomic analysis based on the selective isolation of multiply charged peptides (RH peptides) containing arginine and histidine residues is described. Two pools of proteins are digested in tandem with lysyl-endopeptidase and trypsin and the primary amino groups of proteolytic peptides are separately labeled with d3 - and d0 -acetic anhydride. This reaction has a dual purpose: (i) to allow the relative protein quantification in two different conditions and (ii) to restrict the positive charges of peptides to the presence of arginine and histidine. The N -acylated peptides are separated by cation-exchange chromatography into two groups, neutral and singly charged peptides (R,+,H,,,1) that are neither retained nor analyzed, whereas the multiply charged peptides (R,+,H>1) are retained into the column and can be eluted in batch or further fractionated using a saline gradient before LC-MS/MS analysis. In silico analysis revealed that the selective isolation of RH peptides considerably simplifies the complex mixture of peptides (three RH peptides/protein) and at the same time they represent 84% of the whole proteomes. The selectivity, and recovery of the method were evaluated with model proteins and with a complex mixture of proteins extracted from Vibrio cholerae. [source] Proteomics cataloging analysis of human expressed prostatic secretions reveals rich source of biomarker candidatesPROTEOMICS - CLINICAL APPLICATIONS, Issue 4 2008Runsheng Li Abstract Expressed prostatic secretions (EPS) contain proteins of prostate origin that may reflect the health status of the prostate and be used as diagnostic markers for prostate diseases including prostatitis, benign prostatic hyperplasia, and prostate cancer. Despite their importance and potential applications, a complete catalog of EPS proteins is not yet available. We, therefore, undertook a comprehensive analysis of the EPS proteome using 2-D micro-LC combined with MS/MS. Using stringent filtering criteria, we identified a list of 114 proteins with at least two unique-peptide hits and an additional 75 proteins with only a single unique-peptide hit. The proteins identified include kallikrein 2 (KLK2), KLK3 (prostate-specific antigen), KLK11, and nine cluster of differentiation (CD) molecules including CD10, CD13, CD14, CD26, CD66a, CD66c, CD 143, CD177, and CD224. To our knowledge, this list represents the first comprehensive characterization of the EPS proteome, and it provides a candidate biomarker list for targeted quantitative proteomics analysis using a multiple reaction monitoring (MRM) approach. To help prioritize candidate biomarkers, we constructed a protein,protein interaction network of the EPS proteins using Cytoscape (www.cytoscape.org), and overlaid the expression level changes from the Oncomine database onto the network. [source] | ||||||||||