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At least 433 records · Page 24Linked to original sources
PubMed2006

[Proteome analysis--basis for individualized pancreatic carcinoma therapy?].

Ductal pancreatic adenocarcinoma is a dismal disease, having the worst prognosis of all solid tumors. While genomics and transcriptomics have provided a wealth of data, no contribution has been made to clinical medicine in terms of diagnostic or prognostic markers. Hope lies in yet another novel technology, proteomics. Conceptually, proteomics bears the advantage of incorporating both posttranslational modifications as well as host factors. This is thought to be important in factors influencing survival such as chemoresistance. This tutorial review discusses the state of the art in pancreatic cancer proteomics in light of technical developments. At this moment, proteomics is still at the beginning in clinical application. First results, however, suggest some hope for the development of a new understanding of the molecular biology in pancreatic cancer yielding into very specific markers of disease or allowing a rational and individualized therapy.

Adenocarcinoma↗
PubMed2006

Analysis of the ribosomal protein S19 interactome.

Ribosomal protein S19 (RPS19) is a 16-kDa protein found mainly as a component of the ribosomal 40 S subunit. Its mutations are responsible for Diamond Blackfan anemia, a congenital disease characterized by defective erythroid progenitor maturation. Dysregulation of RPS19 has therefore been implicated in this defective erythropoiesis, although the link between them is still unclear. Two not mutually exclusive hypotheses have been proposed: altered protein synthesis and loss of unknown functions not directly connected with the structural role of RPS19 in the ribosome. A role in rRNA processing has been surmised for the yeast ortholog, whereas the extracellular RPS19 dimer has a monocyte chemotactic activity. Three proteins are known to interact with RPS19: FGF2, complement component 5 receptor 1, and a nucleolar protein called RPS19-binding protein. We have used a yeast two-hybrid approach to identify a fourth protein: the serine-threonine kinase PIM1. The present study describes our use of proteomics strategies to look for proteins interacting with RPS19 to determine its functions. Proteins were isolated by affinity purification with a GST-RPS19 recombinant protein and identified using LCMS/MS analysis coupled to bioinformatics tools. We identified 159 proteins from the following Gene Ontology categories: NTPases (ATPases and GTPases; five proteins), hydrolases/helicases (19 proteins), isomerases (two proteins), kinases (three proteins), splicing factors (five proteins), structural constituents of ribosome (29 proteins), transcription factors (11 proteins), transferases (five proteins), transporters (nine proteins), DNA/RNA-binding protein species (53 proteins), other (one dehydrogenase protein, one ligase protein, one peptidase protein, one receptor protein, and one translation elongation factor), and 13 proteins of still unknown function. Proteomics results were validated by affinity purification and Western blotting. These interactions were further confirmed by co-immunoprecipitation using a monoclonal RPS19 antibody. Many interactors are nucleolar proteins and thus are expected to take part in the RPS19 interactome; however, some proteins suggest additional functional roles for RPS19.

Glutathione Transferase↗
PubMed2005

PIMWalker: visualising protein interaction networks using the HUPO PSI molecular interaction format.

UNLABELLED: This article reports on PIMWalker, a free and interactive tool for visualising protein interaction networks. PIMWalker handles the unified molecular interaction (MI) format defined by members of the Proteomics Standards Initiative (the PSI MI format), and it is thus directly and easily usable by bench biologists. PIMWalker also comes with a documented, open-source Javatrade mark application programming interface allowing the bioinformatic programmer to easily extend the functions. AVAILABILITY: PIMWalker is available under a free license from http://pim.hybrigenics.com/pimwalker.

Computer Graphics↗
PubMed2006

Comparative proteome approach to characterize the high-pressure stress response of Lactobacillus sanfranciscensis DSM 20451(T).

High hydrostatic pressure (HHP) exerts diverse effects on microorganisms, leading to stress response and cell death. While inactivation of microorganisms by lethal HHP is well investigated in the context of food preservation and the hygienic safety of minimal food processes, sublethal HHP stress response and its effect on adaptation and cross-protection is less understood. In this study, the HHP stress response of Lactobacillus sanfranciscensis was characterized and compared with cold, heat, salt, acid and starvation stress at the proteome level by using 2-DE so as to provide insight into general versus specific stress responses. Sixteen proteins were found to be affected by HHP and were identified by using N-terminal amino acid sequencing and MS. Only one slightly increased protein was specific to the HHP response and showed homology to a clp protease. The other proteins were influenced by most of the investigated stresses in a similar way as HHP. The highest similarity in the HHP proteome was found to be with cold- and NaCl-stressed cells, with 11 overlapping proteins. At the proteome level, L. sanfranciscensis appears to use overlapping subsets of stress-inducible proteins rather than stereotype responses. Our data suggest that a specific pressure response does not exist in this bacteria.

Amino Acid Sequence↗
PubMedDate not supplied

Genistein reverses changes of the proteome induced by oxidized-LDL in EA.hy 926 human endothelial cells.

Endothelial cells are primary targets for pro-atherosclerotic stressors such as oxidized LDL (ox-LDL). The isoflavone genistein, on the other hand, is suggested to prevent a variety of processes underlying atherosclerosis and cardiovascular diseases. By analyzing the proteome of EA.hy 926 endothelial cells, here we show, that genistein reverses the ox-LDL-induced changes of the steady-state levels of several proteins involved in atherosclerosis. These alterations caused by genistein are functionally linked to the inhibition of ox-LDL induced apoptosis.

Cell Line↗
PubMed2005

The quest for brain disorder biomarkers.

The identification of disease markers in tissues and body fluids requires an extensive and thorough analysis of its protein constituents. In our efforts to identify biomarkers for affective and neurological disorders we are pursuing several different strategies. On one hand we are using animal models that represent defined phenotypes characteristic for the respective disorder in humans. In addition, we are analyzing human specimens from carefully phenotyped patient groups. Several fractions representing different protein classes from human cerebrospinal fluid obtained by lumbar puncture are used for this purpose. Our biomarker identification efforts range from classical proteomics approaches such as two dimensional gel electrophoresis and mass spectrometry to phage display screens with cerebrospinal fluid antibodies.

Animals↗
PubMed2005

[Comparative proteomics analysis of differentially expressed metastasis-associated proteins in human ovarian cancer cell lines].

OBJECTIVE: To find the key proteins associated with metastasis of ovarian cancer, and find potential diagnostic markers and therapeutic targets of this malignancy. METHODS: A comparative proteomic strategy, in a combination of two-dimensional electrophoresis separation and mass spectrometry identification, was adopted to search for proteome alternations in an ovarian cancer mother cell line HO-8910 and its highly metastatic cell subline HO-8910PM. RESULTS: Twenty-one significantly different spots (two-fold increase or decrease) were detected between the two cell lines, of which 17 candidate proteins were successfully identified and characterized. Compared with those in HO-8910 mother cell line, 16 proteins were significantly up-regulated, while 5 proteins down-regulated in the highly metastatic cell subline HO-8910PM. The seventeen identified proteins could be functionally classified into 7 groups as following: zinc finger protein, calcium-binding protein, DNA repair and synthesis protein, cell regulatory protein, metabolism-related protein, cell surface antigen, cell signals and transducing protein. CONCLUSIONS: The results suggest that an obviously differential proteomic expression exists between the human ovarian cancer mother cell line HO-8910 and highly metastatic cell subline HO-8910PM. It provides a clue for further identification of metastasis-related proteins, novel diagnostic markers as well as therapeutic targets of this malignancy.

Cell Line, Tumor↗
PubMed2005

The wheat (Triticum aestivum L.) leaf proteome.

The wheat leaf proteome was mapped and partially characterized to function as a comparative template for future wheat research. In total, 404 proteins were visualized, and 277 of these were selected for analysis based on reproducibility and relative quantity. Using a combination of protein and expressed sequence tag database searching, 142 proteins were putatively identified with an identification success rate of 51%. The identified proteins were grouped according to their functional annotations with the majority (40%) being involved in energy production, primary, or secondary metabolism. Only 8% of the protein identifications lacked ascertainable functional annotation. The 51% ratio of successful identification and the 8% unclear functional annotation rate are major improvements over most previous plant proteomic studies. This clearly indicates the advancement of the plant protein and nucleic acid sequence and annotation data available in the databases, and shows the enhanced feasibility of future wheat leaf proteome research.

Computational Biology↗
PubMed2006

Increase diagnostic efficacy by combined use of fingerprint markers in mass spectrometry--plasma peptidomes from nasopharyngeal cancer patients for example.

OBJECTIVES: There is no plasma marker for detecting nasopharyngeal cancer (NPC). We developed a bead-based affinity fractionated proteomic method to search potential plasma markers for NPC. DESIGN AND METHODS: Affinity purification of heparinized plasma with Cu-chelated beads and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis were used to screen potential NPC markers. We compiled MS protein profiles for 47 patients with NPC and compared them to profiles from 28 healthy controls. The spectra were statistically analyzed using flexAnalysis and ClinProt bioinformatics software. Diagnostic efficacy was evaluated by determination of the assay sensitivity and specificity of each marker. RESULTS: Twelve mass fingerprint markers differing between cancer and control spectra were found. The sensitivities of these NPC markers are various ranging from 36% to 83%, and the specificities were all over 90%. Combine use of these markers significantly increases diagnostic efficacy. In which, the combined markers (2020 Da and 4635 Da) possess best discriminator with high sensitivity (94%) and specificity (93%). We further identify a C3 fragment, C3f, that may serve as a biomarker for NPC. CONCLUSION: The combined use of mass fingerprint markers in plasma proteome will enhance diagnostic efficacy for NPC. This method can be applied to search for novel plasma markers for cancers.

Adult↗
PubMed2002

High throughput peptide mass fingerprinting and protein macroarray analysis using chemical printing strategies.

We describe a chemical printer that uses piezoelectric pulsing for rapid, accurate, and non-contact microdispensing of fluid for proteomic analysis of immobilized protein macroarrays. We demonstrate protein digestion and peptide mass fingerprinting analysis of human plasma and platelet proteins direct from a membrane surface subsequent to defined microdispensing of trypsin and matrix solutions, hence bypassing multiple liquid-handling steps. Detection of low abundance, alkaline proteins from whole human platelet extracts has been highlighted. Membrane immobilization of protein permits archiving of samples pre-/post-analysis and provides a means for subanalysis using multiple chemistries. This study highlights the ability to increase sequence coverage for protein identification using multiple enzymes and to characterize N-glycosylation modifications using a combination of PNGase F and trypsin. We also demonstrate microdispensing of multiple serum samples in a quantitative microenzyme-linked immunosorbent assay format to rapidly screen protein macroarrays for pathogen-derived antigens. We anticipate the chemical printer will be a major component of proteomic platforms for high throughput protein identification and characterization with widespread applications in biomedical and diagnostic discovery.

Amino Acid Sequence↗
PubMed2007

Proteomics discovery of metalloproteinase substrates in the cellular context by iTRAQ labeling reveals a diverse MMP-2 substrate degradome.

Elucidation of protease substrate degradomes is essential for understanding the function of proteolytic pathways in the protease web and how proteases regulate cell function. We identified matrix metalloproteinase-2 (MMP-2) cleaved proteins, solubilized pericellular matrix, and shed cellular ectodomains in the cellular context using a new multiplex proteomics approach. Tryptic peptides of intact and cleaved proteins, collected from conditioned culture medium of Mmp2(-/-) fibroblasts expressing low levels of transfected active human MMP-2 at different time points, were amine-labeled with iTRAQ mass tags. Peptide identification and relative quantitation between active and inactive protease transfectants were achieved following tag fragmentation during tandem MS. Known substrates of MMP-2 were identified thereby validating this technique with many novel MMP-2 substrates including the CX(3)CL1 chemokine fractalkine, osteopontin, galectin-1, and HSP90alpha also being identified and biochemically confirmed. In comparison with ICAT-labeling and quantitation, 8-9-fold more proteins and substrates were identified by iTRAQ. "Peptide mapping," the location of multiple peptides identified within a particular protein by iTRAQ in combination with their relative abundance ratios, enabled the domain shed and general location of the cleavage site to be identified in the native cellular substrate. Hence this advance in degradomics cell-based screens for native protein substrates casts new light on the roles for proteases in cell function.

Amino Acid Sequence↗
PubMed2001

Characterisation of rice anther proteins expressed at the young microspore stage.

In combination with two-dimensional polyacrylamide gel electrophoresis (2-DE) protein mapping and mass spectrometry analysis, the pattern of gene expression in specific tissues at a specific stage can be displayed and characterised. We used this approach for rice (Oryza sativa L. cultivar Doongara) to display and assign identity to proteins in the anthers at the young microspore stage. Over 4000 anther proteins in the pI range of 4-11 and molecular mass range of 6-122 kDa were reproducibly resolved after silver staining, representing about 10% of the estimated total genomic output of rice. Two hundred and seventy-three protein spots have been extracted either from polyninylidene diffluoride membrane blots or from colloidal Coomassie blue stained 2-DE gels and analysed by N-terminal sequencing, Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MS) analysis or tandem MS sequencing. This enabled identification of 53 anther protein spots representing 43 different proteins. Using the publicly available rice expressed sequence tag (EST) database at the National Centre for Biotechnology Information, a further 37 protein spots were matched to ESTs. After BLAST searching with these ESTs, we were able to predict the identity of 22 of these protein spots. Proteome reference maps of rice anthers have been constructed according to the SWISS-2DPAGE standards and are available for public access at http://semele.anu.edu.au/2d/2d.html.

Electrophoresis, Gel, Two-Dimensional↗
PubMedDate not supplied

Cancer proteomics: from biomarker discovery to signal pathway profiling.

In the post-genomic era of science, the field of proteomics promises the discovery of new molecular targets for therapy, biomarkers for early detection, and new endpoints for therapeutic efficacy and toxicity. Patient-specific targeted therapeutics with reduced toxicity and increased efficacy, the ultimate goal for rational drug development, can only be achieved if direct analyses of the tissue cells in the actual human malignancy are analyzed. To that end, technologies such as Laser Capture Microdissection (LCM), is providing unparalleled access to the purified diseased human cells directly from tissue specimens. However, limited availability of patient material is a challenge towards the development of new highly sensitive proteomic methodologies. Two-dimensional gel electrophoresis (2D-PAGE), still the mainstay of most proteomic analysis of disease, is being complemented, and in some instances replaced by new exciting approaches to multiparametric protein characterization. The use of rapid, high throughput mass spectrometric-based fingerprints of peptides and proteins may prove to be valuable for new molecular classification of human tumors and disease stages. Coupled with LCM, high-density protein arrays, antibody arrays, and small molecular arrays, could have a substantial impact on proteomic profiling of human malignancies. Finally, detailed real-time knowledge about the states of intracellular signaling circuitry and pathways in the normal and malignant cells before, during and after therapy will yield invaluable information about mechanism of action and efficacy of existing and novel therapeutics for the treatment of human cancer.

Biomarkers↗
PubMed2006

Identification of S-nitrosylation motifs by site-specific mapping of the S-nitrosocysteine proteome in human vascular smooth muscle cells.

S-nitrosylation, the selective modification of cysteine residues in proteins to form S-nitrosocysteine, is a major emerging mechanism by which nitric oxide acts as a signaling molecule. Even though nitric oxide is intimately involved in the regulation of vascular smooth muscle cell functions, the potential protein targets for nitric oxide modification as well as structural features that underlie the specificity of protein S-nitrosocysteine formation in these cells remain unknown. Therefore, we used a proteomic approach using selective peptide capturing and site-specific adduct mapping to identify the targets of S-nitrosylation in human aortic smooth muscle cells upon exposure to S-nitrosocysteine and propylamine propylamine NONOate. This strategy identified 20 unique S-nitrosocysteine-containing peptides belonging to 18 proteins including cytoskeletal proteins, chaperones, proteins of the translational machinery, vesicular transport, and signaling. Sequence analysis of the S-nitrosocysteine-containing peptides revealed the presence of acid/base motifs, as well as hydrophobic motifs surrounding the identified cysteine residues. High-resolution immunogold electron microscopy supported the cellular localization of several of these proteins. Interestingly, seven of the 18 proteins identified are localized within the ER/Golgi complex, suggesting a role for S-nitrosylation in membrane trafficking and ER stress response in vascular smooth muscle.

Amino Acid Sequence↗
PubMed2005

Proteomic analysis of differentially expressed proteins between the male and female worm of Schistosoma japonicum after pairing.

Identification of differentially expressed proteins between the male and female worm of Schistosoma japonicum may provide new insights into the development of schistosomes, especially the molecular mechanism of female worm maturation induced by the male worm after pairing. Comparative two-dimensional gel electrophoresis (2-DE) and mass spectrometry were employed to separate and identify differentially expressed proteins between the male and female worm after pairing. Soluble and hydrophobic proteins from egg, schistosomulum (14 days), and female and male worms at adult stage (42 days) were separated by a sequential extraction method followed by 2-DE and 2-DE images were constructed. There were 1016 +/- 67, 1808 +/- 89, 1142 +/- 45 and 1288 +/- 32 spots detected for soluble proteins and 1425 +/- 108, 952 +/- 59, 847 +/- 75 and 965 +/- 69 spots for hydrophobic proteins from egg, schistosomulum, and adult stage female and male worms, respectively. The differentially and uniquely expressed proteins from male and female worms after pairing (42 days) include 41 +/- 4 and 23 +/- 2 unique spots for soluble proteins, and 11 +/- 1 and 26 +/- 3 unique spots for hydrophobic proteins, respectively. Matrix-assisted laser desorption/ionization-time of flight and electrospray ionization-tandem mass spectrometry were employed to analyze 12 unique spots from the female worm and 16 unique spots from the male worm for peptide mass fingerprinting and sequencing. The results showed that the main functions of these differentially expressed proteins were in signal transduction, metabolism and transcriptional regulation etc. Comparison of the schistosomes proteome between male and female worms may permit the identification of protein candidates for the development of vaccines or new targets for drug development against schistosomiasis.

Animals↗
PubMed2006

Proteome analysis of resting human neutrophils.

Neutrophils constitute the first line of host defense against pathogens. In the present study 2-D gel electrophoresis-mass spectrometry technology was employed to analyze the human resting neutrophils proteome. One hundred and two conserved spots were subjected to peptide mass fingerprinting, yielding 22 identifications. Among the identified proteins, nine are related to the inflammatory process, two polypeptides are assigned to metabolic functions and five are classified as structural.

Adult↗
PubMed2004

Proteomic analysis of azole resistance in Candida albicans clinical isolates.

Changes in protein expression within a matched set of Candida albicans isolates representing the acquisition of azole resistance were examined by two-dimensional polyacrylamide gel electrophoresis and peptide mass fingerprinting. Proteins differentially expressed in association with azole resistance included Grp2p, Ifd1p, Ifd4p, Ifd5p, and Erg10p, a protein involved in the ergosterol biosynthesis pathway.

Amino Acids, Sulfur↗
PubMed2004

Predicting co-complexed protein pairs using genomic and proteomic data integration.

BACKGROUND: Identifying all protein-protein interactions in an organism is a major objective of proteomics. A related goal is to know which protein pairs are present in the same protein complex. High-throughput methods such as yeast two-hybrid (Y2H) and affinity purification coupled with mass spectrometry (APMS) have been used to detect interacting proteins on a genomic scale. However, both Y2H and APMS methods have substantial false-positive rates. Aside from high-throughput interaction screens, other gene- or protein-pair characteristics may also be informative of physical interaction. Therefore it is desirable to integrate multiple datasets and utilize their different predictive value for more accurate prediction of co-complexed relationship. RESULTS: Using a supervised machine learning approach--probabilistic decision tree, we integrated high-throughput protein interaction datasets and other gene- and protein-pair characteristics to predict co-complexed pairs (CCP) of proteins. Our predictions proved more sensitive and specific than predictions based on Y2H or APMS methods alone or in combination. Among the top predictions not annotated as CCPs in our reference set (obtained from the MIPS complex catalogue), a significant fraction was found to physically interact according to a separate database (YPD, Yeast Proteome Database), and the remaining predictions may potentially represent unknown CCPs. CONCLUSIONS: We demonstrated that the probabilistic decision tree approach can be successfully used to predict co-complexed protein (CCP) pairs from other characteristics. Our top-scoring CCP predictions provide testable hypotheses for experimental validation.

Computational Biology↗