Proteomic Analysis of Human Tendon and Ligament: Solubilization and Analysis of Insoluble Extracellular Matrix in Connective Tissues.

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Sato, Nori; Taniguchi, Takako; Goda, Yuichiro; Kosaka, Hirofumi; Higashino, Kosaku; Sakai, Toshinori; Katoh, Shinsuke; Yasui, Natsuo; Sairyo, Koichi; Taniguchi, Hisaaki

2016-12-02

Connective tissues such as tendon, ligament and cartilage are mostly composed of extracellular matrix (ECM). These tissues are insoluble, mainly due to the highly cross-linked ECM proteins such as collagens. Difficulties obtaining suitable samples for mass spectrometric analysis render the application of modern proteomic technologies difficult. Complete solubilization of them would not only elucidate protein composition of normal tissues but also reveal pathophysiology of pathological tissues. Here we report complete solubilization of human Achilles tendon and yellow ligament, which is achieved by chemical digestion combined with successive protease treatment including elastase. The digestion mixture was subjected to liquid chromatography-mass spectrometry. The low specificity of elastase was overcome by accurate mass analysis achieved using FT-ICR-MS. In addition to the detailed proteome of both tissues, we also quantitatively determine the major protein composition of samples, by measuring peak area of some characteristic peptides detected in tissue samples and in purified proteins. As a result, differences between human Achilles tendon and yellow ligament were elucidated at molecular level.

Comparative Proteomics of Human Monkeypox and Vaccinia Intracellular Mature and Extracellular Enveloped Virions

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Manes, Nathan P.; Estep, Ryan D.; Mottaz, Heather M.; Moore, Ronald J.; Clauss, Therese RW; Monroe, Matthew E.; Du, Xiuxia; Adkins, Joshua N.; Wong, Scott; Smith, Richard D.

2008-03-07

Orthopoxviruses are the largest and most complex of the animal viruses. In response to the recent emergence of monkeypox in Africa and the threat of smallpox bioterrorism, virulent (monkeypox virus) and benign (vaccinia virus) orthopoxviruses were proteomically compared with the goal of identifying proteins required for pathogenesis. Orthopoxviruses were grown in HeLa cells to two different viral forms (intracellular mature virus and extracellular enveloped virus), purified by sucrose gradient ultracentrifugation, denatured using RapiGest™ surfactant, and digested with trypsin. Unfractionated samples and strong cation exchange HPLC fractions were analyzed by reversed-phase LC-MS/MS, and analyses of the MS/MS spectra using SEQUEST® and X! Tandem resulted in the identification of hundreds of monkeypox, vaccinia, and copurified host proteins. The unfractionated samples were additionally analyzed by LC-MS on an LTQ-Orbitrap™, and the accurate mass and elution time tag approach was used to perform quantitative comparisons. Possible pathophysiological roles of differentially expressed orthopoxvirus genes are discussed.

Proteomic analysis of extracellular proteins from Aspergillus oryzae grown under submerged and solid-state culture conditions.

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Oda, Ken; Kakizono, Dararat; Yamada, Osamu; Iefuji, Haruyuki; Akita, Osamu; Iwashita, Kazuhiro

2006-05-01

Filamentous fungi are widely used for the production of homologous and heterologous proteins. Recently, there has been increasing interest in Aspergillus oryzae because of its ability to produce heterologous proteins in solid-state culture. To provide an overview of protein secretion by A. oryzae in solid-state culture, we carried out a comparative proteome analysis of extracellular proteins in solid-state and submerged (liquid) cultures. Extracellular proteins prepared from both cultures sequentially from 0 to 40 h were subjected to two-dimensional electrophoresis, and protein spots at 40 h were identified by peptide mass fingerprinting using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. We also attempted to identify cell wall-bound proteins of the submerged culture. We analyzed 85 spots from the solid-state culture and 110 spots from the submerged culture. We identified a total of 29 proteins, which were classified into 4 groups. Group 1 consisted of extracellular proteins specifically produced in the solid-state growth condition, such as glucoamylase B and alanyl dipeptidyl peptidase. Group 2 consisted of extracellular proteins specifically produced in the submerged condition, such as glucoamylase A (GlaA) and xylanase G2 (XynG2). Group 3 consisted of proteins produced in both conditions, such as xylanase G1. Group 4 consisted of proteins that were secreted to the medium in the solid-state growth condition but trapped in the cell wall in the submerged condition, such as alpha-amylase (TAA) and beta-glucosidase (Bgl). A Northern analysis of seven genes from the four groups suggested that the secretion of TAA and Bgl was regulated by trapping these proteins in the cell wall in submerged culture and that secretion of GlaA and XynG2 was regulated at the posttranscriptional level in the solid-state culture.

Circulating extracellular vesicles with specific proteome and liver microRNAs are potential biomarkers for liver injury in experimental fatty liver disease.

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Davide Povero

Full Text Available Nonalcoholic fatty liver disease (NAFLD is the most common chronic liver disease in both adult and children. Currently there are no reliable methods to determine disease severity, monitor disease progression, or efficacy of therapy, other than an invasive liver biopsy.Choline Deficient L-Amino Acid (CDAA and high fat diets were used as physiologically relevant mouse models of NAFLD. Circulating extracellular vesicles were isolated, fully characterized by proteomics and molecular analyses and compared to control groups. Liver-related microRNAs were isolated from purified extracellular vesicles and liver specimens.We observed statistically significant differences in the level of extracellular vesicles (EVs in liver and blood between two control groups and NAFLD animals. Time-course studies showed that EV levels increase early during disease development and reflect changes in liver histolopathology. EV levels correlated with hepatocyte cell death (r2 = 0.64, p<0.05, fibrosis (r2 = 0.66, p<0.05 and pathological angiogenesis (r2 = 0.71, p<0.05. Extensive characterization of blood EVs identified both microparticles (MPs and exosomes (EXO present in blood of NAFLD animals. Proteomic analysis of blood EVs detected various differentially expressed proteins in NAFLD versus control animals. Moreover, unsupervised hierarchical clustering identified a signature that allowed for discrimination between NAFLD and controls. Finally, the liver appears to be an important source of circulating EVs in NAFLD animals as evidenced by the enrichment in blood with miR-122 and 192--two microRNAs previously described in chronic liver diseases, coupled with a corresponding decrease in expression of these microRNAs in the liver.These findings suggest a potential for using specific circulating EVs as sensitive and specific biomarkers for the noninvasive diagnosis and monitoring of NAFLD.

Extracellular glycosylphosphatidylinositol-anchored mannoproteins and proteases of Cryptococcus neoformans.

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Eigenheer, Richard A; Jin Lee, Young; Blumwald, Eduardo; Phinney, Brett S; Gelli, Angie

2007-06-01

Extracellular proteins of Cryptococcus neoformans are involved in the pathogenesis of cryptococcosis, and some are immunoreactive antigens that may potentially serve as candidates for vaccine development. To further study the extracellular proteome of the human fungal pathogen Cry. neoformans, we conducted a proteomic analysis of secreted and cell wall-bound proteins with an acapsular strain of Cry. neoformans. Proteins were identified from both intact cells and cell walls. In both cases, extracellular proteins were removed with trypsin or beta-glucanase, and then all proteins/peptides were purified by solid-phase extraction, spin dialysis, and HPLC, and identified by liquid chromatography-mass spectrometry. This study identified 29 extracellular proteins with a predicted N-terminal signal sequence and also a predicted glycosylphosphatidylinositol anchor motif in more than half. Among the novel proteins identified were five glycosylphosphatidylinositol-anchored proteins with extensive Ser/Thr-rich regions but no apparent functional domains, a glycosylphosphatidylinositol-anchored aspartic protease, and a metalloprotease with structural similarity to an elastinolytic metalloprotease of Aspergillus fumigatus. This study suggests that Cry. neoformans has the machinery required to target glycosylphosphatidylinositol-anchored proteins to the cell wall, and it confirms the extracellular proteolytic ability of Cry. neoformans.

Characterization of yeast extracellular vesicles: evidence for the participation of different pathways of cellular traffic in vesicle biogenesis.

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Débora L Oliveira

2010-06-01

Full Text Available Extracellular vesicles in yeast cells are involved in the molecular traffic across the cell wall. In yeast pathogens, these vesicles have been implicated in the transport of proteins, lipids, polysaccharide and pigments to the extracellular space. Cellular pathways required for the biogenesis of yeast extracellular vesicles are largely unknown.We characterized extracellular vesicle production in wild type (WT and mutant strains of the model yeast Saccharomyces cerevisiae using transmission electron microscopy in combination with light scattering analysis, lipid extraction and proteomics. WT cells and mutants with defective expression of Sec4p, a secretory vesicle-associated Rab GTPase essential for Golgi-derived exocytosis, or Snf7p, which is involved in multivesicular body (MVB formation, were analyzed in parallel. Bilayered vesicles with diameters at the 100-300 nm range were found in extracellular fractions from yeast cultures. Proteomic analysis of vesicular fractions from the cells aforementioned and additional mutants with defects in conventional secretion pathways (sec1-1, fusion of Golgi-derived exocytic vesicles with the plasma membrane; bos1-1, vesicle targeting to the Golgi complex or MVB functionality (vps23, late endosomal trafficking revealed a complex and interrelated protein collection. Semi-quantitative analysis of protein abundance revealed that mutations in both MVB- and Golgi-derived pathways affected the composition of yeast extracellular vesicles, but none abrogated vesicle production. Lipid analysis revealed that mutants with defects in Golgi-related components of the secretory pathway had slower vesicle release kinetics, as inferred from intracellular accumulation of sterols and reduced detection of these lipids in vesicle fractions in comparison with WT cells.Our results suggest that both conventional and unconventional pathways of secretion are required for biogenesis of extracellular vesicles, which demonstrate the

Neutrophil Extracellular Traps in Ulcerative Colitis

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Bjerg Bennike, Tue; Carlsen, Thomas Gelsing; Ellingsen, Torkell

2015-01-01

microscopy and confocal microscopy. RESULTS: We identified and quantified 5711 different proteins with proteomics. The abundance of the proteins calprotectin and lactotransferrin in the tissue correlated with the degree of tissue inflammation as determined by histology. However, fecal calprotectin did...... not correlate. Forty-six proteins were measured with a statistically significant differences in abundances between the UC colon tissue and controls. Eleven of the proteins with increased abundances in the UC biopsies were associated with neutrophils and neutrophil extracellular traps. The findings were...... validated by microscopy, where an increased abundance of neutrophils and the presence of neutrophil extracellular traps by extracellular DNA present in the UC colon tissue were confirmed. CONCLUSIONS: Neutrophils, induced neutrophil extracellular traps, and several proteins that play a part in innate...

Proteomics of Fuchs' Endothelial Corneal Dystrophy support that the extracellular matrix of Descemet's membrane is disordered

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Poulsen, Ebbe Toftgaard; Dyrlund, Thomas F; Runager, Kasper

2014-01-01

Fuchs' endothelial corneal dystrophy (FECD) is a major corneal disorder affecting the innermost part of the cornea, leading to visual impairment. As the morphological changes in FECD are mainly observed in the extracellular matrix of the Descemet's membrane/endothelial layer we determined...... that the morphological changes observed in FECD is caused in part by an aberrant assembly of the extracellular matrix within the Descemet's membrane/endothelial layer......., respectively, of which 10 were significantly regulated. The results indicated that the level of type VIII collagen was unaltered even though the protein previously has been implicated in familial early onset forms of the disease. Using the second relative quantitation method iTRAQ we identified 22...

The external face of Candida albicans: A proteomic view of the cell surface and the extracellular environment.

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Gil-Bona, Ana; Amador-García, Ahinara; Gil, Concha; Monteoliva, Lucia

2018-05-30

The cell surface and secreted proteins are the initial points of contact between Candida albicans and the host. Improvements in protein extraction approaches and mass spectrometers have allowed researchers to obtain a comprehensive knowledge of these external subproteomes. In this paper, we review the published proteomic studies that have examined C. albicans extracellular proteins, including the cell surface proteins or surfome and the secreted proteins or secretome. The use of different approaches to isolate cell wall and cell surface proteins, such as fractionation approaches or cell shaving, have resulted in different outcomes. Proteins with N-terminal signal peptide, known as classically secreted proteins, and those that lack the signal peptide, known as unconventionally secreted proteins, have been consistently identified. Existing studies on C. albicans extracellular vesicles reveal that they are relevant as an unconventional pathway of protein secretion and can help explain the presence of proteins without a signal peptide, including some moonlighting proteins, in the cell wall and the extracellular environment. According to the global view presented in this review, cell wall proteins, virulence factors such as adhesins or hydrolytic enzymes, metabolic enzymes and stress related-proteins are important groups of proteins in C. albicans surfome and secretome. Candida albicans extracellular proteins are involved in biofilm formation, cell nutrient acquisition and cell wall integrity maintenance. Furthermore, these proteins include virulence factors and immunogenic proteins. This review is of outstanding interest, not only because it extends knowledge of the C. albicans surface and extracellular proteins that could be related with pathogenesis, but also because it presents insights that may facilitate the future development of new antifungal drugs and vaccines and contributes to efforts to identify new biomarkers that can be employed to diagnose candidiasis

Proteomic approaches in cancer risk and response assessment.

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Petricoin, Emanuel F; Liotta, Lance A

2004-02-01

Proteomics is more than just a list-generating exercise where increases or decreases in protein expression are identified. Proteomic technologies will ultimately characterize information-flow through the protein circuitry that interconnects the extracellular microenvironment to the serum or plasma macroenvironment through intracellular signaling systems and their control of gene transcription. The nature of this information can be a cause or a consequence of disease processes and how patients respond to therapy. Analysis of human cancer as a model for how proteomics can have an impact at the bedside can take advantage of several promising new proteomic technologies. These technologies are being developed for early detection and risk assessment, therapeutic targeting and patient-tailored therapy.

ISDoT: in situ decellularization of tissues for high-resolution imaging and proteomic analysis of native extracellular matrix

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Mayorca-Guiliani, Alejandro E.; Madsen, Chris D.; Cox, Thomas R.

2017-01-01

The extracellular matrix (ECM) is a master regulator of cellular phenotype and behavior. It has a crucial role in both normal tissue homeostasis and disease pathology. Here we present a fast and efficient approach to enhance the study of ECM composition and structure. Termed in situ...... decellularization of tissues (ISDoT), it allows whole organs to be decellularized, leaving native ECM architecture intact. These three-dimensional decellularized tissues can be studied using high-resolution fluorescence and second harmonic imaging, and can be used for quantitative proteomic interrogation of the ECM....... Our method is superior to other methods tested in its ability to preserve the structural integrity of the ECM, facilitate high-resolution imaging and quantitatively detect ECM proteins. In particular, we performed high-resolution sub-micron imaging of matrix topography in normal tissue and over...

Proteomics study of extracellular fibrinolytic proteases from Bacillus licheniformis RO3 and Bacillus pumilus 2.g isolated from Indonesian fermented food

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Nur Afifah, Diana; Rustanti, Ninik; Anjani, Gemala; Syah, Dahrul; Yanti; Suhartono, Maggy T.

2017-02-01

This paper presents the proteomics study which includes separation, identification and characterization of proteins. The experiment on Indonesian fermented food such as extracellular fibrinolytic protease from Bacillus licheniformis RO3 and Bacillus pumilus 2.g isolated from red oncom and tempeh gembus was conducted. The experimental works comprise the following steps: (1) a combination of one- and two-dimensional electrophoresis analysis, (2) mass spectrometry analysis using MALDI-TOF-MS and (3) investigation using protein database. The result suggested that there were new two protein fractions of B. licheniformis RO3 and three protein fractions of B. pumilus 2.g. These result has not been previously reported.

Proteomics of Plant Pathogenic Fungi

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Raquel González-Fernández

2010-01-01

Full Text Available Plant pathogenic fungi cause important yield losses in crops. In order to develop efficient and environmental friendly crop protection strategies, molecular studies of the fungal biological cycle, virulence factors, and interaction with its host are necessary. For that reason, several approaches have been performed using both classical genetic, cell biology, and biochemistry and the modern, holistic, and high-throughput, omic techniques. This work briefly overviews the tools available for studying Plant Pathogenic Fungi and is amply focused on MS-based Proteomics analysis, based on original papers published up to December 2009. At a methodological level, different steps in a proteomic workflow experiment are discussed. Separate sections are devoted to fungal descriptive (intracellular, subcellular, extracellular and differential expression proteomics and interactomics. From the work published we can conclude that Proteomics, in combination with other techniques, constitutes a powerful tool for providing important information about pathogenicity and virulence factors, thus opening up new possibilities for crop disease diagnosis and crop protection.

Comparative proteomic analysis of normal and collagen IX null mouse cartilage reveals altered extracellular matrix composition and novel components of the collagen IX interactome.

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Brachvogel, Bent; Zaucke, Frank; Dave, Keyur; Norris, Emma L; Stermann, Jacek; Dayakli, Münire; Koch, Manuel; Gorman, Jeffrey J; Bateman, John F; Wilson, Richard

2013-05-10

Collagen IX is an integral cartilage extracellular matrix component important in skeletal development and joint function. Proteomic analysis and validation studies revealed novel alterations in collagen IX null cartilage. Matrilin-4, collagen XII, thrombospondin-4, fibronectin, βig-h3, and epiphycan are components of the in vivo collagen IX interactome. We applied a proteomics approach to advance our understanding of collagen IX ablation in cartilage. The cartilage extracellular matrix is essential for endochondral bone development and joint function. In addition to the major aggrecan/collagen II framework, the interacting complex of collagen IX, matrilin-3, and cartilage oligomeric matrix protein (COMP) is essential for cartilage matrix stability, as mutations in Col9a1, Col9a2, Col9a3, Comp, and Matn3 genes cause multiple epiphyseal dysplasia, in which patients develop early onset osteoarthritis. In mice, collagen IX ablation results in severely disturbed growth plate organization, hypocellular regions, and abnormal chondrocyte shape. This abnormal differentiation is likely to involve altered cell-matrix interactions but the mechanism is not known. To investigate the molecular basis of the collagen IX null phenotype we analyzed global differences in protein abundance between wild-type and knock-out femoral head cartilage by capillary HPLC tandem mass spectrometry. We identified 297 proteins in 3-day cartilage and 397 proteins in 21-day cartilage. Components that were differentially abundant between wild-type and collagen IX-deficient cartilage included 15 extracellular matrix proteins. Collagen IX ablation was associated with dramatically reduced COMP and matrilin-3, consistent with known interactions. Matrilin-1, matrilin-4, epiphycan, and thrombospondin-4 levels were reduced in collagen IX null cartilage, providing the first in vivo evidence for these proteins belonging to the collagen IX interactome. Thrombospondin-4 expression was reduced at the mRNA level

The diversity and specificity of the extracellular proteome in the cellulolytic bacterium Caldicellulosiruptor bescii is driven by the nature of the cellulosic growth substrate.

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Poudel, Suresh; Giannone, Richard J; Basen, Mirko; Nookaew, Intawat; Poole, Farris L; Kelly, Robert M; Adams, Michael W W; Hettich, Robert L

2018-01-01

Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates. Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs), ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. This study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono

Proteomic analysis of rutin-induced secreted proteins from Aspergillus flavus.

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Medina, Martha L; Kiernan, Urban A; Francisco, Wilson A

2004-03-01

Few studies have been conducted to identify the extracellular proteins and enzymes secreted by filamentous fungi, particularly with respect to dispensable metabolic pathways. Proteomic analysis has proven to be the most powerful method for identification of proteins in complex mixtures and is suitable for the study of the alteration of protein expression under different environmental conditions. The filamentous fungus Aspergillus flavus can degrade the flavonoid rutin as the only source of carbon via an extracellular enzyme system. In this study, a proteomic analysis was used to differentiate and identify the extracellular rutin-induced and non-induced proteins secreted by A. flavus. The secreted proteins were analyzed by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. While 15 rutin-induced proteins and 7 non-induced proteins were identified, more than 90 protein spots remain unidentified, indicating that these proteins are either novel proteins or proteins that have not yet been sequenced.

Proteomic analysis of pig (Sus scrofa olfactory soluble proteome reveals O-GlcNAcylation of secreted odorant-binding proteins

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Patricia eNAGNAN-LE MEILLOUR

2014-12-01

Full Text Available The diversity of olfactory binding proteins (OBPs is a key point to understand their role in molecular olfaction. Since only few different sequences were characterized in each mammalian species, they have been considered as passive carriers of odors and pheromones. We have explored the soluble proteome of pig nasal mucus, taking benefit of the powerful tools of proteomics. Combining two-dimensional electrophoresis, mass spectrometry and western-blot with specific antibodies, our analyses revealed for the first time that the pig nasal mucus is mainly composed of secreted OBP isoforms, some of them being potentially modified by O-GlcNAcylation. An ortholog gene of the glycosyltransferase responsible of the O-GlcNAc linking on extracellular proteins in Drosophila and Mouse (EOGT was amplified from tissues of pigs of different ages and sex. The sequence was used in a phylogenetic analysis, which evidenced conservation of EOGT in insect and mammalian models studied in molecular olfaction. Extracellular O-GlcNAcylation of secreted OBPs could finely modulate their binding specificities to odors and pheromones. This constitutes a new mechanism for extracellular signaling by OBPs, suggesting that they act as the first step of odor discrimination.

Effect of greenhouse conditions on the leaf apoplastic proteome of Coffea arabica plants.

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Guerra-Guimarães, Leonor; Vieira, Ana; Chaves, Inês; Pinheiro, Carla; Queiroz, Vagner; Renaut, Jenny; Ricardo, Cândido P

2014-06-02

This work describes the coffee leaf apoplastic proteome and its modulation by the greenhouse conditions. The apoplastic fluid (APF) was obtained by leaf vacuum infiltration, and the recovered proteins were separated by 2-DE and subsequently identified by matrix assisted laser desorption/ionization time of flight-mass spectrometry, followed by homology search in EST coffee databases. Prediction tools revealed that the majority of the 195 identified proteins are involved in cell wall metabolism and in stress/defense responses. Although most of the proteins follow the classical secretory mechanism, a low percentage of them seem to result from unconventional secretion (leaderless secreted proteins). Principal components analysis revealed that the APF samples formed two distinct groups, with the temperature amplitude mostly contributing for this separation (higher or lower than 10°C, respectively). Sixty one polypeptide spots allowed defining these two groups and 28 proteins were identified, belonging to carbohydrate metabolism, cell wall modification and proteolysis. Interestingly stress/defense proteins appeared as more abundant in Group I which is associated with a higher temperature amplitude. It seems that the proteins in the coffee leaf APF might be implicated in structural modifications in the extracellular space that are crucial for plant development/adaptation to the conditions of the prevailing environment. This is the first detailed proteomic study of the coffee leaf apoplastic fluid (APF) and of its modulation by the greenhouse conditions. The comprehensive overview of the most abundant proteins present in the extra-cellular compartment is particularly important for the understanding of coffee responses to abiotic/biotic stress. This article is part of a Special Issue entitled: Environmental and structural proteomics. Copyright © 2014 Elsevier B.V. All rights reserved.

Differential secretome analysis of Pseudomonas syringae pv tomato using gel-free MS proteomics

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Jörg eSchumacher

2014-07-01

Full Text Available The plant pathogen Pseudomonas syringae pv. tomato (DC3000 causes virulence by delivering effector proteins into host plant cells through its type three secretion system (T3SS. In response to the plant environment DC3000 expresses hypersensitive response and pathogenicity genes (hrp. Pathogenesis depends on the ability of the pathogen to manipulate the plant metabolism and to inhibit plant immunity, which depends to a large degree on the plant’s capacity to recognise both pathogen and microbial determinants (PAMP/MAMP-triggered immunity. We have developed and employed MS-based shotgun and targeted proteomics to (i elucidate the extracellular and secretome composition of DC3000 and (ii evaluate temporal features of the assembly of the T3SS and the secretion process together with its dependence of pH. The proteomic screen, under hrp inducing in vitro conditions, of extracellular and cytoplasmatic fractions indicated the segregated presence of not only T3SS implicated proteins such as HopK1, HrpK1, HrpA1 and Avrpto1, but also of proteins not usually associated with the T3SS or with pathogenicity. Using multiple reaction monitoring MS (MRM-MS to quantify HrpA1 and Avrpto1, we found that HrpA1 is rapidly expressed, at a strict pH-dependent rate and is post-translationally processed extracellularly. These features appear to not interfere with rapid Avrpto1 expression and secretion but may suggest some temporal post-translational regulatory mechanism of the T3SS assembly. The high specificity and sensitivity of the MRM-MS approach should provide a powerful tool to measure secretion and translocation in infected tissues.

Mass Spectrometry for Translational Proteomics: Progress and Clinical Implications

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Baker, Erin Shammel; Liu, Tao; Petyuk, Vladislav A.; Burnum-Johnson, Kristin E.; Ibrahim, Yehia M.; Anderson, Gordon A.; Smith, Richard D.

2012-08-31

Mass spectrometry (MS)-based proteomics measurements have become increasingly utilized in a wide range of biological and biomedical applications, and have significantly enhanced the understanding of the complex and dynamic nature of the proteome and its connections to biology and diseases. While some MS techniques such as those for targeted analysis are increasingly applied with great success, others such as global quantitative analysis (for e.g. biomarker discovery) are more challenging and continue to be developed and refined to provide the desired throughput, sensitivity and/ or specificity. New MS capabilities and proteomics-based pipelines/strategies also keep enhancing for the advancement of clinical proteomics applications such as protein biomarker discovery and validation. Herein, we provide a brief review to summarize the current state of MS-based proteomics with respect to its advantages and present limitations, while highlighting its potential in future clinical applications.

Functional proteomic of Matrix Metallo-proteinases (MMP) dedicated to the detection of active forms of MMP in complex proteome

International Nuclear Information System (INIS)

David, A.

2007-07-01

The Matrix Metallo-proteinases (M.M.P.) represent a family of Zinc dependent extracellular proteinases able to cleave collectively all the proteins constituting the extracellular matrix. Currently, 23 human M.M.P. have been identified and are characterized by their sequence in amino-acids and their highly conserved 3 D structure. These enzymes are expressed constitutively during the tissue remodeling process. Their over-expression in various diseases tightly related to inflammatory processes (arthritis, emphysema, cancer) described M.M.P. as choice therapeutic targets. However, as the tissue remodeling implicates modification of cellular contacts, M.M.P. appear currently as proteins involved in signalling pathways. Recent works demonstrating that M.M.P. are able to cleave substrates, which are different than proteins constituting the extracellular matrix, reinforce this vision. In order to identify the individual role and the protein expression level of M.M.P. in pathological context, we developed a new technique of functional proteomics dedicated to the detection of active forms of M.M.P. in tumour samples. This technique relied on the development of a new photoaffinity probe, based on the structure of a potent phosphinic inhibitor of M.M.P., allowing targeting and isolating active forms of M.M.P. by photoaffinity labelling. Furthermore, as the new developed probe incorporated a radioactive element, photoaffinity labelling permitted to radiolabel the targeted proteins. This probe demonstrated in vitro its remarkable ability to covalently modify the h M.M.P.-12, with a singular cross-linking yield, determined at 42 %, displaying an extremely sensitive detection (2.5 fmoles of h M.M.P.-12). When added to complex proteome, the photoaffinity probe presents the same sensibility of detection for the h M.M.P.-12 (5 fmoles); importantly, in this case, h M.M.P.-12 represents only 0.001 % of the totality of the proteins present in the sample. Moreover, this technique allows

Organized proteomic heterogeneity in colorectal cancer liver metastases and implications for therapies.

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Turtoi, Andrei; Blomme, Arnaud; Debois, Delphine; Somja, Joan; Delvaux, David; Patsos, Georgios; Di Valentin, Emmanuel; Peulen, Olivier; Mutijima, Eugène Nzaramba; De Pauw, Edwin; Delvenne, Philippe; Detry, Olivier; Castronovo, Vincent

2014-03-01

Tumor heterogeneity is a major obstacle for developing effective anticancer treatments. Recent studies have pointed to large stochastic genetic heterogeneity within cancer lesions, where no pattern seems to exist that would enable a more structured targeted therapy approach. Because to date no similar information is available at the protein (phenotype) level, we employed matrix assisted laser desorption ionization (MALDI) image-guided proteomics and explored the heterogeneity of extracellular and membrane subproteome in a unique collection of eight fresh human colorectal carcinoma (CRC) liver metastases. Monitoring the spatial distribution of over 1,000 proteins, we found unexpectedly that all liver metastasis lesions displayed a reproducible, zonally delineated pattern of functional and therapeutic biomarker heterogeneity. The peritumoral region featured elevated lipid metabolism and protein synthesis, the rim of the metastasis displayed increased cellular growth, movement, and drug metabolism, whereas the center of the lesion was characterized by elevated carbohydrate metabolism and DNA-repair activity. From the aspect of therapeutic targeting, zonal expression of known and novel biomarkers was evident, reinforcing the need to select several targets in order to achieve optimal coverage of the lesion. Finally, we highlight two novel antigens, LTBP2 and TGFBI, whose expression is a consistent feature of CRC liver metastasis. We demonstrate their in vivo antibody-based targeting and highlight their potential usefulness for clinical applications. The proteome heterogeneity of human CRC liver metastases has a distinct, organized pattern. This particular hallmark can now be used as part of the strategy for developing rational therapies based on multiple sets of targetable antigens. © 2014 by the American Association for the Study of Liver Diseases.

Cross-sample validation provides enhanced proteome coverage in rat vocal fold mucosa.

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Nathan V Welham

2011-03-01

Full Text Available The vocal fold mucosa is a biomechanically unique tissue comprised of a densely cellular epithelium, superficial to an extracellular matrix (ECM-rich lamina propria. Such ECM-rich tissues are challenging to analyze using proteomic assays, primarily due to extensive crosslinking and glycosylation of the majority of high M(r ECM proteins. In this study, we implemented an LC-MS/MS-based strategy to characterize the rat vocal fold mucosa proteome. Our sample preparation protocol successfully solubilized both proteins and certain high M(r glycoconjugates and resulted in the identification of hundreds of mucosal proteins. A straightforward approach to the treatment of protein identifications attributed to single peptide hits allowed the retention of potentially important low abundance identifications (validated by a cross-sample match and de novo interpretation of relevant spectra while still eliminating potentially spurious identifications (global single peptide hits with no cross-sample match. The resulting vocal fold mucosa proteome was characterized by a wide range of cellular and extracellular proteins spanning 12 functional categories.

Proteome reference map of Drosophila melanogaster head.

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Lee, Tian-Ren; Huang, Shun-Hong; Lee, Chi-Ching; Lee, Hsiao-Yun; Chan, Hsin-Tzu; Lin, Kuo-Sen; Chan, Hong-Lin; Lyu, Ping-Chiang

2012-06-01

Drosophila melanogaster has been used as a genetic model organism to understand the fundamental molecular mechanisms in human biology including memory formation that has been reported involving protein synthesis and/or post-translational modification. In this study, we employed a proteomic platform based on fluorescent 2DE and MALDI-TOF MS to build a standard D. melanogaster head proteome map for proteome-proteome comparison. In order to facilitate the comparison, an interactive database has been constructed for systematically integrating and analyzing the proteomes from different conditions and further implicated to study human diseases related to D. melanogaster model. In summary, the fundamental head proteomic database and bioinformatic analysis will be useful for further elucidating the biological mechanisms such as memory formation and neurodegenerative diseases. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The extracellular proteome of Bifidobacterium animalis subsp. lactis BB‐12 reveals proteins with putative roles in probiotic effects

DEFF Research Database (Denmark)

Gilad, Ofir; Svensson, Birte; Viborg, Alexander Holm

2011-01-01

Probiotics are live microorganisms that exert health‐promoting effects on the human host, as demonstrated for numerous strains of the genus Bifidobacterium. To unravel the proteins involved in the interactions between the host and the extensively used and well‐studied probiotic strain Bifidobacte......Probiotics are live microorganisms that exert health‐promoting effects on the human host, as demonstrated for numerous strains of the genus Bifidobacterium. To unravel the proteins involved in the interactions between the host and the extensively used and well‐studied probiotic strain...... Bifidobacterium animalis subsp. lactis BB‐12, proteins secreted by the bacterium, i.e. belonging to the extracellular proteome present in the culture medium, were identified by 2‐DE coupled with MALDI‐TOF MS. Among the 74 distinct proteins identified, 31 are predicted to carry out their physiological role either...... functions include binding of plasminogen, formation of fimbriae, adhesion to collagen, attachment to mucin and intestinal cells as well as induction of immunomodulative response. These findings suggest a role of the proteins in colonization of the gastrointestinal tract, adhesion to host tissues...

Triple SILAC quantitative proteomic analysis reveals differential abundance of cell signaling proteins between normal and lung cancer-derived exosomes.

Science.gov (United States)

Clark, David J; Fondrie, William E; Yang, Austin; Mao, Li

2016-02-05

Exosomes are 30-100 nm sized membrane vesicles released by cells into the extracellular space that mediate intercellular communication via transfer of proteins and other biological molecules. To better understand the role of these microvesicles in lung carcinogenesis, we employed a Triple SILAC quantitative proteomic strategy to examine the differential protein abundance between exosomes derived from an immortalized normal bronchial epithelial cell line and two non-small cell lung cancer (NSCLC) cell lines harboring distinct activating mutations in the cell signaling molecules: Kirsten rat sarcoma viral oncogene homolog (KRAS) or epidermal growth factor receptor (EGFR). In total, we were able to quantify 721 exosomal proteins derived from the three cell lines. Proteins associated with signal transduction, including EGFR, GRB2 and SRC, were enriched in NSCLC exosomes, and could actively regulate cell proliferation in recipient cells. This study's investigation of the NSCLC exosomal proteome has identified enriched protein cargo that can contribute to lung cancer progression, which may have potential clinical implications in biomarker development for patients with NSCLC. The high mortality associated with lung cancer is a result of late-stage diagnosis of the disease. Current screening techniques used for early detection of lung cancer lack the specificity for accurate diagnosis. Exosomes are nano-sized extracellular vesicles, and the increased abundance of select protein cargo in exosomes derived from cancer cells may be used for diagnostic purposes. In this paper, we applied quantitative proteomic analysis to elucidate abundance differences in exosomal protein cargo between two NSCLC cell lines with distinctive oncogene mutations and an immortalized normal bronchial epithelial cell line. This study revealed proteins associated with cell adhesion, the extracellular matrix, and a variety of signaling molecules were enriched in NSCLC exosomes. The present data reveals

Proteomic analysis of temperature dependent extracellular proteins from Aspergillus fumigatus grown under solid-state culture condition.

Science.gov (United States)

Adav, Sunil S; Ravindran, Anita; Sze, Siu Kwan

2013-06-07

Fungal species of the genus Aspergillus are filamentous ubiquitous saprophytes that play a major role in lignocellulosic biomass recycling and also are considered as cell factories for the production of organic acids, pharmaceuticals, and industrially important enzymes. Analysis of extracellular secreted biomass degrading enzymes using complex lignocellulosic biomass as a substrate by solid-state fermentation could be a more practical approach to evaluate application of the enzymes for lignocellulosic biorefinery. This study isolated a fungal strain from compost, identified as Aspergillus fumigatus, and further analyzed it for lignocellulolytic enzymes at different temperatures using label free quantitative proteomics. The profile of secretome composition discovered cellulases, hemicellulases, lignin degrading proteins, peptidases and proteases, and transport and hypothetical proteins; while protein abundances and further their hierarchical clustering analysis revealed temperature dependent expression of these enzymes during solid-state fermentation of sawdust. The enzyme activities and protein abundances as determined by exponentially modified protein abundance index (emPAI) indicated the maximum activities at the range of 40-50 °C, demonstrating the thermophilic nature of the isolate A. fumigatus LF9. Characterization of the thermostability of secretome suggested the potential of the isolated fungal strain in the production of thermophilic biomass degrading enzymes for industrial application.

Analysis of extracellular proteins of Aspergillus oryzae grown on soy sauce koji.

Science.gov (United States)

Liang, Yanchang; Pan, Li; Lin, Ying

2009-01-01

Aspergillus oryzae AS 3.951 is widely used in Chinese soy sauce manufacture, but little is known about the profiles of the extracellular proteins from the culture of soybean koji. In this study, we carried out MALDI-TOF/TOF MS analysis of extracellular proteins during koji culture. Besides well-known proteins (TAA and Oryzin), a variety of aminopeptidase and proteases were identical at the proteome level. This suggests that A. oryzae AS 3.951 has a powerful capacity to digest soybean protein.

The urinary proteome in diabetes and diabetes-associated complications

DEFF Research Database (Denmark)

Rossing, Kasper; Mischak, Harald; Rossing, Peter

2008-01-01

Diabetes represents one of the main chronic diseases worldwide. Diabetes and its associated complications may be detectable even at early stages in the urinary proteome. In this article we review the current literature on urinary proteomics applied to the study of diabetes and diabetic...... complications. Further, we present recent data that strongly indicate urinary proteome analysis may be a valuable tool in detecting diabetes-associated pathophysiological changes at an early stage, and also may enable assessment of disease progression and efficacy of therapy. Current data indicate that collagen......-derived peptides represent one of the main peptidic components in urine, which are consistently found at reduced levels in diabetes. It is tempting to speculate that this decrease in urinary collagen-derived peptides is related to an increase in extracellular matrix deposition which is a major complication...

Comparative proteomic analysis of extracellular proteins expressed by various clonal types of Staphylococcus aureus and during planktonic growth and biofilm development.

Science.gov (United States)

Atshan, Salman S; Shamsudin, Mariana N; Sekawi, Zamberi; Thian Lung, Leslie T; Barantalab, Fatemeh; Liew, Yun K; Alreshidi, Mateg Ali; Abduljaleel, Salwa A; Hamat, Rukman A

2015-01-01

Staphylococcus aureus is well known for its biofilm formation with rapid emergence of new clones circulating worldwide. The main objectives of the study were (1) to identify possible differences in protein expression among various and closely related clonal types of S. aureus, (2) to establish the differences in protein expression in terms of size of protein spots and its intensities between bacteria which are grown statically (biofilm formation) with that of under aeration and agitation, and (3) to compare the differences in protein expression as a function of time (in hours). In this study, we selected six clinical isolates comprising two similar (MRSA-527 and MRSA-524) and four different (MRSA-139, MSSA-12E, MSSA-22d, and MSSA-10E) types identified by spa typing, MLST and SCCmec typing. We performed 2D gel migration comparison. Also, two MRSA isolates (527 and 139) were selected to determine quantitative changes in the level of extracellular proteins at different biofilm growth time points of 12, 24, and 48 h. The study was done using a strategy that combines 2-DGE and LC-MS/MS analysis for absolute quantification and identification of the extracellular proteins. The 2DGE revealed that the proteomic profiles for the isolates belonging to the similar spa, MLST, and SCCmec types were still quite different. Among the extracellular proteins secreted at different time points of biofilm formation, significant changes in protein expression were observed at 48 h incubation as compared to the exponential growth at 12 h incubation. The main conclusion of the work is that the authors do observe differences among isolates, and growth conditions do influence the protein content at different time points of biofilm formation.

Quantitative Proteomics Reveals Temporal Proteomic Changes in Signaling Pathways during BV2 Mouse Microglial Cell Activation.

Science.gov (United States)

Woo, Jongmin; Han, Dohyun; Wang, Joseph Injae; Park, Joonho; Kim, Hyunsoo; Kim, Youngsoo

2017-09-01

The development of systematic proteomic quantification techniques in systems biology research has enabled one to perform an in-depth analysis of cellular systems. We have developed a systematic proteomic approach that encompasses the spectrum from global to targeted analysis on a single platform. We have applied this technique to an activated microglia cell system to examine changes in the intracellular and extracellular proteomes. Microglia become activated when their homeostatic microenvironment is disrupted. There are varying degrees of microglial activation, and we chose to focus on the proinflammatory reactive state that is induced by exposure to such stimuli as lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). Using an improved shotgun proteomics approach, we identified 5497 proteins in the whole-cell proteome and 4938 proteins in the secretome that were associated with the activation of BV2 mouse microglia by LPS or IFN-γ. Of the differentially expressed proteins in stimulated microglia, we classified pathways that were related to immune-inflammatory responses and metabolism. Our label-free parallel reaction monitoring (PRM) approach made it possible to comprehensively measure the hyper-multiplex quantitative value of each protein by high-resolution mass spectrometry. Over 450 peptides that corresponded to pathway proteins and direct or indirect interactors via the STRING database were quantified by label-free PRM in a single run. Moreover, we performed a longitudinal quantification of secreted proteins during microglial activation, in which neurotoxic molecules that mediate neuronal cell loss in the brain are released. These data suggest that latent pathways that are associated with neurodegenerative diseases can be discovered by constructing and analyzing a pathway network model of proteins. Furthermore, this systematic quantification platform has tremendous potential for applications in large-scale targeted analyses. The proteomics data for

SELDI-TOF-based serum proteomic pattern diagnostics for early detection of cancer.

Science.gov (United States)

Petricoin, Emanuel F; Liotta, Lance A

2004-02-01

Proteomics is more than just generating lists of proteins that increase or decrease in expression as a cause or consequence of pathology. The goal should be to characterize the information flow through the intercellular protein circuitry that communicates with the extracellular microenvironment and then ultimately to the serum/plasma macroenvironment. The nature of this information can be a cause, or a consequence, of disease and toxicity-based processes. Serum proteomic pattern diagnostics is a new type of proteomic platform in which patterns of proteomic signatures from high dimensional mass spectrometry data are used as a diagnostic classifier. This approach has recently shown tremendous promise in the detection of early-stage cancers. The biomarkers found by SELDI-TOF-based pattern recognition analysis are mostly low molecular weight fragments produced at the specific tumor microenvironment.

The proteome of human saliva

Science.gov (United States)

Griffin, Timothy J.

2013-05-01

Human saliva holds tremendous potential for transforming disease and health diagnostics given its richness of molecular information and non-invasive collection. Enumerating its molecular constituents is an important first step towards reaching this potential. Among the molecules in saliva, proteins and peptides arguably have the most value: they can directly indicate biochemical functions linked to a health condition/disease state, and they are attractive targets for biomarker assay development. However, cataloging and defining the human salivary proteome is challenging given the dynamic, chemically heterogeneous and complex nature of the system. In addition, the overall human saliva proteome is composed of several "sub-proteomes" which include: intact full length proteins, proteins carrying post-translational modifications (PTMs), low molecular weight peptides, and the metaproteome, derived from protein products from nonhuman organisms (e.g. microbes) present in the oral cavity. Presented here will be a summary of communal efforts to meet the challenge of characterizing the multifaceted saliva proteome, focusing on the use of mass spectrometry as the proteomic technology of choice. Implications of these efforts to characterize the salivary proteome in the context of disease diagnostics will also be discussed.

Noninvasive diagnosis of intraamniotic infection: proteomic biomarkers in vaginal fluid.

Science.gov (United States)

Hitti, Jane; Lapidus, Jodi A; Lu, Xinfang; Reddy, Ashok P; Jacob, Thomas; Dasari, Surendra; Eschenbach, David A; Gravett, Michael G; Nagalla, Srinivasa R

2010-07-01

We analyzed the vaginal fluid proteome to identify biomarkers of intraamniotic infection among women in preterm labor. Proteome analysis was performed on vaginal fluid specimens from women with preterm labor, using multidimensional liquid chromatography, tandem mass spectrometry, and label-free quantification. Enzyme immunoassays were used to quantify candidate proteins. Classification accuracy for intraamniotic infection (positive amniotic fluid bacterial culture and/or interleukin-6 >2 ng/mL) was evaluated using receiver-operator characteristic curves obtained by logistic regression. Of 170 subjects, 30 (18%) had intraamniotic infection. Vaginal fluid proteome analysis revealed 338 unique proteins. Label-free quantification identified 15 proteins differentially expressed in intraamniotic infection, including acute-phase reactants, immune modulators, high-abundance amniotic fluid proteins and extracellular matrix-signaling factors; these findings were confirmed by enzyme immunoassay. A multi-analyte algorithm showed accurate classification of intraamniotic infection. Vaginal fluid proteome analyses identified proteins capable of discriminating between patients with and without intraamniotic infection. Copyright (c) 2010 Mosby, Inc. All rights reserved.

Quantitative proteomic analysis of extracellular matrix extracted from mono- and dual-species biofilms of Fusobacterium nucleatum and Porphyromonas gingivalis.

Science.gov (United States)

Mohammed, Marwan Mansoor Ali; Pettersen, Veronika Kuchařová; Nerland, Audun H; Wiker, Harald G; Bakken, Vidar

2017-04-01

The Gram-negative bacteria Fusobacterium nucleatum and Porphyromonas gingivalis are members of a complex dental biofilm associated with periodontal disease. In this study, we cultured F. nucleatum and P. gingivalis as mono- and dual-species biofilms, and analyzed the protein composition of the biofilms extracellular polymeric matrix (EPM) by high-resolution liquid chromatography-tandem mass spectrometry. Label-free quantitative proteomic analysis was used for identification of proteins and sequence-based functional characterization for their classification and prediction of possible roles in EPM. We identified 542, 93 and 280 proteins in the matrix of F. nucleatum, P. gingivalis, and the dual-species biofilm, respectively. Nearly 70% of all EPM proteins in the dual-species biofilm originated from F. nucleatum, and a majority of these were cytoplasmic proteins, suggesting an enhanced lysis of F. nucleatum cells. The proteomic analysis also indicated an interaction between the two species: 22 F. nucleatum proteins showed differential levels between the mono and dual-species EPMs, and 11 proteins (8 and 3 from F. nucleatum and P. gingivalis, respectively) were exclusively detected in the dual-species EPM. Oxidoreductases and chaperones were among the most abundant proteins identified in all three EPMs. The biofilm matrices in addition contained several known and hypothetical virulence proteins, which can mediate adhesion to the host cells and disintegration of the periodontal tissues. This study demonstrated that the biofilm matrix of two important periodontal pathogens consists of a multitude of proteins whose amounts and functionalities vary largely. Relatively high levels of several of the detected proteins might facilitate their potential use as targets for the inhibition of biofilm development. Copyright © 2017 Elsevier Ltd. All rights reserved.

Comparative Proteomic Analysis of Supportive and Unsupportive Extracellular Matrix Substrates for Human Embryonic Stem Cell Maintenance*

Science.gov (United States)

Soteriou, Despina; Iskender, Banu; Byron, Adam; Humphries, Jonathan D.; Borg-Bartolo, Simon; Haddock, Marie-Claire; Baxter, Melissa A.; Knight, David; Humphries, Martin J.; Kimber, Susan J.

2013-01-01

Human embryonic stem cells (hESCs) are pluripotent cells that have indefinite replicative potential and the ability to differentiate into derivatives of all three germ layers. hESCs are conventionally grown on mitotically inactivated mouse embryonic fibroblasts (MEFs) or feeder cells of human origin. In addition, feeder-free culture systems can be used to support hESCs, in which the adhesive substrate plays a key role in the regulation of stem cell self-renewal or differentiation. Extracellular matrix (ECM) components define the microenvironment of the niche for many types of stem cells, but their role in the maintenance of hESCs remains poorly understood. We used a proteomic approach to characterize in detail the composition and interaction networks of ECMs that support the growth of self-renewing hESCs. Whereas many ECM components were produced by supportive and unsupportive MEF and human placental stromal fibroblast feeder cells, some proteins were only expressed in supportive ECM, suggestive of a role in the maintenance of pluripotency. We show that identified candidate molecules can support attachment and self-renewal of hESCs alone (fibrillin-1) or in combination with fibronectin (perlecan, fibulin-2), in the absence of feeder cells. Together, these data highlight the importance of specific ECM interactions in the regulation of hESC phenotype and provide a resource for future studies of hESC self-renewal. PMID:23658023

Functional proteomic of Matrix Metallo-proteinases (MMP) dedicated to the detection of active forms of MMP in complex proteome; Proteomique fonctionnelle dediee aux Metalloproteases Matricelles (MMPs): developpement d'une methode extremement sensible permettant la detection des formes actives des MMPs dans des proteomes complexes

Energy Technology Data Exchange (ETDEWEB)

David, A

2007-07-15

The Matrix Metallo-proteinases (M.M.P.) represent a family of Zinc dependent extracellular proteinases able to cleave collectively all the proteins constituting the extracellular matrix. Currently, 23 human M.M.P. have been identified and are characterized by their sequence in amino-acids and their highly conserved 3 D structure. These enzymes are expressed constitutively during the tissue remodeling process. Their over-expression in various diseases tightly related to inflammatory processes (arthritis, emphysema, cancer) described M.M.P. as choice therapeutic targets. However, as the tissue remodeling implicates modification of cellular contacts, M.M.P. appear currently as proteins involved in signalling pathways. Recent works demonstrating that M.M.P. are able to cleave substrates, which are different than proteins constituting the extracellular matrix, reinforce this vision. In order to identify the individual role and the protein expression level of M.M.P. in pathological context, we developed a new technique of functional proteomics dedicated to the detection of active forms of M.M.P. in tumour samples. This technique relied on the development of a new photoaffinity probe, based on the structure of a potent phosphinic inhibitor of M.M.P., allowing targeting and isolating active forms of M.M.P. by photoaffinity labelling. Furthermore, as the new developed probe incorporated a radioactive element, photoaffinity labelling permitted to radiolabel the targeted proteins. This probe demonstrated in vitro its remarkable ability to covalently modify the h M.M.P.-12, with a singular cross-linking yield, determined at 42 %, displaying an extremely sensitive detection (2.5 fmoles of h M.M.P.-12). When added to complex proteome, the photoaffinity probe presents the same sensibility of detection for the h M.M.P.-12 (5 fmoles); importantly, in this case, h M.M.P.-12 represents only 0.001 % of the totality of the proteins present in the sample. Moreover, this technique allows

Functional proteomic of Matrix Metallo-proteinases (MMP) dedicated to the detection of active forms of MMP in complex proteome; Proteomique fonctionnelle dediee aux Metalloproteases Matricelles (MMPs): developpement d'une methode extremement sensible permettant la detection des formes actives des MMPs dans des proteomes complexes

Energy Technology Data Exchange (ETDEWEB)

David, A

2007-07-15

The Matrix Metallo-proteinases (M.M.P.) represent a family of Zinc dependent extracellular proteinases able to cleave collectively all the proteins constituting the extracellular matrix. Currently, 23 human M.M.P. have been identified and are characterized by their sequence in amino-acids and their highly conserved 3 D structure. These enzymes are expressed constitutively during the tissue remodeling process. Their over-expression in various diseases tightly related to inflammatory processes (arthritis, emphysema, cancer) described M.M.P. as choice therapeutic targets. However, as the tissue remodeling implicates modification of cellular contacts, M.M.P. appear currently as proteins involved in signalling pathways. Recent works demonstrating that M.M.P. are able to cleave substrates, which are different than proteins constituting the extracellular matrix, reinforce this vision. In order to identify the individual role and the protein expression level of M.M.P. in pathological context, we developed a new technique of functional proteomics dedicated to the detection of active forms of M.M.P. in tumour samples. This technique relied on the development of a new photoaffinity probe, based on the structure of a potent phosphinic inhibitor of M.M.P., allowing targeting and isolating active forms of M.M.P. by photoaffinity labelling. Furthermore, as the new developed probe incorporated a radioactive element, photoaffinity labelling permitted to radiolabel the targeted proteins. This probe demonstrated in vitro its remarkable ability to covalently modify the h M.M.P.-12, with a singular cross-linking yield, determined at 42 %, displaying an extremely sensitive detection (2.5 fmoles of h M.M.P.-12). When added to complex proteome, the photoaffinity probe presents the same sensibility of detection for the h M.M.P.-12 (5 fmoles); importantly, in this case, h M.M.P.-12 represents only 0.001 % of the totality of the proteins present in the sample. Moreover, this technique allows

A Review: Proteomics in Retinal Artery Occlusion, Retinal Vein Occlusion, Diabetic Retinopathy and Acquired Macular Disorders.

Science.gov (United States)

Cehofski, Lasse Jørgensen; Honoré, Bent; Vorum, Henrik

2017-04-28

Retinal artery occlusion (RAO), retinal vein occlusion (RVO), diabetic retinopathy (DR) and age-related macular degeneration (AMD) are frequent ocular diseases with potentially sight-threatening outcomes. In the present review we discuss major findings of proteomic studies of RAO, RVO, DR and AMD, including an overview of ocular proteome changes associated with anti-vascular endothelial growth factor (VEGF) treatments. Despite the severe outcomes of RAO, the proteome of the disease remains largely unstudied. There is also limited knowledge about the proteome of RVO, but proteomic studies suggest that RVO is associated with remodeling of the extracellular matrix and adhesion processes. Proteomic studies of DR have resulted in the identification of potential therapeutic targets such as carbonic anhydrase-I. Proliferative diabetic retinopathy is the most intensively studied stage of DR. Proteomic studies have established VEGF, pigment epithelium-derived factor (PEDF) and complement components as key factors associated with AMD. The aim of this review is to highlight the major milestones in proteomics in RAO, RVO, DR and AMD. Through large-scale protein analyses, proteomics is bringing new important insights into these complex pathological conditions.

Clinical proteomics: Applications for prostate cancer biomarker discovery and detection.

Science.gov (United States)

Petricoin, Emanuel F; Ornstein, David K; Liotta, Lance A

2004-01-01

The science of proteomics comprises much more than simply generating lists of proteins that change in expression as a cause of or consequence of pathophysiology. The goal of proteomics should be to characterize the information flow through the intercellular protein circuitry that communicates with the extracellular microenvironment and then ultimately to the serum/plasma macroenvironment. Serum proteomic pattern diagnostics is a new type of proteomic concept in which patterns of ion signatures generated from high dimensional mass spectrometry data are used as diagnostic classifiers. This recent approach has exciting potential for clinical utility of diagnostic patterns because low molecular weight metabolites, peptides, and protein fragments may have higher accuracy than traditional biomarkers of cancer detection. Intriguingly, we now have discovered that this diagnostic information exists in a bound state, complexed with circulating highly abundant carrier proteins. These diagnostic fragments may one day be harvested by circulating nanoparticles, designed to absorb, enrich, and amplify the repertoire of diagnostic biomarkers generated-even at the critical, initial stages of carcinogenesis. Copyright 2004 Elsevier Inc.

Proteomic profiling of Bifidobacterium bifidum S17 cultivated under in vitro conditions

Directory of Open Access Journals (Sweden)

Xiao eWei

2016-02-01

Full Text Available Bifidobacteria are frequently used in probiotic food and dairy products. Bifidobacterium bifidum S17 is a promising probiotic candidate strain that displays strong adhesion to intestinal epithelial cells and elicits potent anti-inflammatory capacity both in vitro and in murine models of colitis. The recently sequenced genome of B. bifidum S17 has a size of about 2.2 Mb and encodes 1,782 predicted protein-coding genes. In the present study, a comprehensive proteomic profiling was carried out to identify and characterize proteins expressed by B. bifidum S17. A total of 1148 proteins entries were identified by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS, representing 64.4% of the predicted proteome. 719 proteins could be assigned to functional categories according to cluster of orthologous groups of proteins (COGs. The COG distribution of the detected proteins highly correlates with that of the complete predicted proteome suggesting a good coverage and representation of the genomic content of B. bifidum S17 by the proteome. COGs that were highly present in the proteome of B. bifidum S17 were Translation, Amino Acid Transport and Metabolism, and Carbohydrate Transport and Metabolism. Complete sets of enzymes for both the bifidus shunt and the Embden-Meyerhof pathway were identified. Further bioinformatic analysis yielded 28 proteins with a predicted extracellular localization including 14 proteins with an LPxTG-motif for cell wall anchoring and two proteins (elongation factor Tu and enolase with a potential moonlighting function in adhesion. Amongst the predicted extracellular proteins were five of six pilin proteins encoded in the B. bifidum S17 genome as well as several other proteins with a potential role in interaction with host structures. The presented results are the first compilation of a proteomic reference profile for a B. bifidum strain and will facilitate analysis of the molecular mechanisms of physiology, host

The landscape of viral proteomics and its potential to impact human health

Energy Technology Data Exchange (ETDEWEB)

Oxford, Kristie L.; Wendler, Jason P.; McDermott, Jason E.; White III, Richard A.; Powell, Joshua D.; Jacobs, Jon M.; Adkins, Joshua N.; Waters, Katrina M.

2016-05-06

Translating the intimate discourse between viruses and their host cells during infection is a challenging but critical task for development of antiviral interventions and diagnostics. Viruses commandeer cellular processes at every step of their life cycle, altering expression of genes and proteins. Advances in mass spectrometry-based proteomic technologies are enhancing studies of viral pathogenesis by identifying virus-induced changes in the protein repertoire of infected cells or extracellular fluids. Interpretation of proteomics results using knowledge of cellular pathways and networks leads to identification of proteins that influence a range of infection processes, thereby focusing efforts for clinical diagnoses and therapeutics development. Herein we discuss applications of global proteomic studies of viral infections with the goal of providing a basis for improved studies that will benefit community-wide data integration and interpretation.

Developmental distribution of the plasma membrane-enriched proteome in the maize primary root growth zone

Directory of Open Access Journals (Sweden)

Zhe eZhang

2013-03-01

Full Text Available Within the growth zone of the maize primary root, there are well-defined patterns of spatial and temporal organization of cell division and elongation. However, the processes underlying this organization remain poorly understood. To gain additional insights into the differences amongst the defined regions, we performed a proteomic analysis focusing on fractions enriched for plasma membrane (PM proteins. The PM is the interface between the plant cell and the apoplast and/or extracellular space. As such, it is a key structure involved in the exchange of nutrients and other molecules as well as in the integration of signals that regulate growth and development. Despite the important functions of PM-localized proteins in mediating these processes, a full understanding of dynamic changes in PM proteomes is often impeded by low relative concentrations relative to total proteins. Using a relatively simple strategy of treating microsomal fractions with Brij-58 detergent to enrich for PM proteins, we compared the developmental distribution of proteins within the root growth zone which revealed a number of previously known as well as novel proteins with interesting patterns of abundance. For instance, the quantitative proteomic analysis detected a gradient of PM aquaporin proteins similar to that previously reported using immunoblot analyses, confirming the veracity of this strategy. Cellulose synthases increased in abundance with increasing distance from the root apex, consistent with expected locations of cell wall deposition. The similar distribution pattern for Brittle-stalk-2-like protein 3 implicate that this protein may also have cell wall related functions. These results show that the simplified PM enrichment method previously demonstrated in Arabidopsis can be successfully applied to completely unrelated plant tissues and provide insights into differences in the PM proteome throughout growth and development zones of the maize primary root.

Developmental distribution of the plasma membrane-enriched proteome in the maize primary root growth zone.

Science.gov (United States)

Zhang, Zhe; Voothuluru, Priyamvada; Yamaguchi, Mineo; Sharp, Robert E; Peck, Scott C

2013-01-01

Within the growth zone of the maize primary root, there are well-defined patterns of spatial and temporal organization of cell division and elongation. However, the processes underlying this organization remain poorly understood. To gain additional insights into the differences amongst the defined regions, we performed a proteomic analysis focusing on fractions enriched for plasma membrane (PM) proteins. The PM is the interface between the plant cell and the apoplast and/or extracellular space. As such, it is a key structure involved in the exchange of nutrients and other molecules as well as in the integration of signals that regulate growth and development. Despite the important functions of PM-localized proteins in mediating these processes, a full understanding of dynamic changes in PM proteomes is often impeded by low relative concentrations relative to total proteins. Using a relatively simple strategy of treating microsomal fractions with Brij-58 detergent to enrich for PM proteins, we compared the developmental distribution of proteins within the root growth zone which revealed a number of previously known as well as novel proteins with interesting patterns of abundance. For instance, the quantitative proteomic analysis detected a gradient of PM aquaporin proteins similar to that previously reported using immunoblot analyses, confirming the veracity of this strategy. Cellulose synthases increased in abundance with increasing distance from the root apex, consistent with expected locations of cell wall deposition. The similar distribution pattern for Brittle-stalk-2-like protein implicates that this protein may also have cell wall related functions. These results show that the simplified PM enrichment method previously demonstrated in Arabidopsis can be successfully applied to completely unrelated plant tissues and provide insights into differences in the PM proteome throughout growth and development zones of the maize primary root.

Methodologies and Perspectives of Proteomics Applied to Filamentous Fungi: From Sample Preparation to Secretome Analysis

Science.gov (United States)

Bianco, Linda; Perrotta, Gaetano

2015-01-01

Filamentous fungi possess the extraordinary ability to digest complex biomasses and mineralize numerous xenobiotics, as consequence of their aptitude to sensing the environment and regulating their intra and extra cellular proteins, producing drastic changes in proteome and secretome composition. Recent advancement in proteomic technologies offers an exciting opportunity to reveal the fluctuations of fungal proteins and enzymes, responsible for their metabolic adaptation to a large variety of environmental conditions. Here, an overview of the most commonly used proteomic strategies will be provided; this paper will range from sample preparation to gel-free and gel-based proteomics, discussing pros and cons of each mentioned state-of-the-art technique. The main focus will be kept on filamentous fungi. Due to the biotechnological relevance of lignocellulose degrading fungi, special attention will be finally given to their extracellular proteome, or secretome. Secreted proteins and enzymes will be discussed in relation to their involvement in bio-based processes, such as biomass deconstruction and mycoremediation. PMID:25775160

Methodologies and perspectives of proteomics applied to filamentous fungi: from sample preparation to secretome analysis.

Science.gov (United States)

Bianco, Linda; Perrotta, Gaetano

2015-03-12

Filamentous fungi possess the extraordinary ability to digest complex biomasses and mineralize numerous xenobiotics, as consequence of their aptitude to sensing the environment and regulating their intra and extra cellular proteins, producing drastic changes in proteome and secretome composition. Recent advancement in proteomic technologies offers an exciting opportunity to reveal the fluctuations of fungal proteins and enzymes, responsible for their metabolic adaptation to a large variety of environmental conditions. Here, an overview of the most commonly used proteomic strategies will be provided; this paper will range from sample preparation to gel-free and gel-based proteomics, discussing pros and cons of each mentioned state-of-the-art technique. The main focus will be kept on filamentous fungi. Due to the biotechnological relevance of lignocellulose degrading fungi, special attention will be finally given to their extracellular proteome, or secretome. Secreted proteins and enzymes will be discussed in relation to their involvement in bio-based processes, such as biomass deconstruction and mycoremediation.

Isolation and characterization of urinary extracellular vesicles: implications for biomarker discovery.

Science.gov (United States)

Merchant, Michael L; Rood, Ilse M; Deegens, Jeroen K J; Klein, Jon B

2017-12-01

Urine is a valuable diagnostic medium and, with the discovery of urinary extracellular vesicles, is viewed as a dynamic bioactive fluid. Extracellular vesicles are lipid-enclosed structures that can be classified into three categories: exosomes, microvesicles (or ectosomes) and apoptotic bodies. This classification is based on the mechanisms by which membrane vesicles are formed: fusion of multivesicular bodies with the plasma membranes (exosomes), budding of vesicles directly from the plasma membrane (microvesicles) or those shed from dying cells (apoptotic bodies). During their formation, urinary extracellular vesicles incorporate various cell-specific components (proteins, lipids and nucleic acids) that can be transferred to target cells. The rigour needed for comparative studies has fueled the search for optimal approaches for their isolation, purification, and characterization. RNA, the newest extracellular vesicle component to be discovered, has received substantial attention as an extracellular vesicle therapeutic, and compelling evidence suggests that ex vivo manipulation of microRNA composition may have uses in the treatment of kidney disorders. The results of these studies are building the case that urinary extracellular vesicles act as mediators of renal pathophysiology. As the field of extracellular vesicle studies is burgeoning, this Review focuses on primary data obtained from studies of human urine rather than on data from studies of laboratory animals or cultured immortalized cells.

Extracellular matrix disruption is an early event in the pathogenesis of skeletal disease in mucopolysaccharidosis I.

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Heppner, Jonathan M; Zaucke, Frank; Clarke, Lorne A

2015-02-01

Progressive skeletal and connective tissue disease represents a significant clinical burden in all of the mucopolysaccharidoses. Despite the introduction of enzyme replacement strategies for many of the mucopolysaccharidoses, symptomatology related to bone and joint disease appears to be recalcitrant to current therapies. In order to address these unmet medical needs a clearer understanding of skeletal and connective tissue disease pathogenesis is required. Historically the pathogenesis of the mucopolysaccharidoses has been assumed to directly relate to progressive storage of glycosaminoglycans. It is now apparent for many lysosomal storage disorders that more complex pathogenic mechanisms underlie patients' clinical symptoms. We have used proteomic and genome wide expression studies in the murine mucopolysaccharidosis I model to identify early pathogenic events occurring in micro-dissected growth plate tissue. Studies were conducted using 3 and 5-week-old mice thus representing a time at which no obvious morphological changes of bone or joints have taken place. An unbiased iTRAQ differential proteomic approach was used to identify candidates followed by validation with multiple reaction monitoring mass spectrometry and immunohistochemistry. These studies reveal significant decreases in six key structural and signaling extracellular matrix proteins; biglycan, fibromodulin, PRELP, type I collagen, lactotransferrin, and SERPINF1. Genome-wide expression studies in embryonic day 13.5 limb cartilage and 5 week growth plate cartilage followed by specific gene candidate qPCR studies in the 5week growth plate identified fourteen significantly deregulated mRNAs (Adamts12, Aspn, Chad, Col2a1, Col9a1, Hapln4, Lum, Matn1, Mmp3, Ogn, Omd, P4ha2, Prelp, and Rab32). The involvement of biglycan, PRELP and fibromodulin; all members of the small leucine repeat proteoglycan family is intriguing, as this protein family is implicated in the pathogenesis of late onset osteoarthritis

Alterations in the Cerebral Microvascular Proteome Expression Profile After Transient Global Cerebral Ischemia in Rat

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Spray, Stine; Johansson, Sara E; Edwards, Alistair V G

2017-01-01

. The proteomic profile of the isolated cerebral microvasculature 72 h after GCI (compared to sham) indicated that the main expressional changes could be divided into nine categories: (1) cellular respiration, (2) remodelling of the extracellular matrix, (3) decreased contractile phenotype, (4) clathrin...

Extraction and Characterization of Extracellular Proteins and Their Post-Translational Modifications from Arabidopsis thaliana Suspension Cell Cultures and Seedlings: A Critical Review

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Mina Ghahremani

2016-09-01

Full Text Available Proteins secreted by plant cells into the extracellular space, consisting of the cell wall, apoplastic fluid, and rhizosphere, play crucial roles during development, nutrient acquisition, and stress acclimation. However, isolating the full range of secreted proteins has proven difficult, and new strategies are constantly evolving to increase the number of proteins that can be detected and identified. In addition, the dynamic nature of the extracellular proteome presents the further challenge of identifying and characterizing the post-translational modifications (PTMs of secreted proteins, particularly glycosylation and phosphorylation. Such PTMs are common and important regulatory modifications of proteins, playing a key role in many biological processes. This review explores the most recent methods in isolating and characterizing the plant extracellular proteome with a focus on the model plant Arabidopsis thaliana, highlighting the current challenges yet to be overcome. Moreover, the crucial role of protein PTMs in cell wall signalling, development, and plant responses to biotic and abiotic stress is discussed.

Elucidation of cross-species proteomic effects in human and hominin bone proteome identification through a bioinformatics experiment.

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Welker, F

2018-02-20

between moderately divergent proteomes, as indicated by almost complete recovery of informative positions in the search against the chimpanzee proteome (≈90%, 6-8 Ma). This provides a bioinformatic background to future phylogenetic and proteomic analysis of ancient hominin proteomes, including the future description of novel hominin amino acid sequences, but also has negative implications for the study of fast-evolving proteins in hominins, non-hominin animals, and ancient bacterial proteins in evolutionary contexts.

Characterization of the canine urinary proteome.

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Brandt, Laura E; Ehrhart, E J; Scherman, Hataichanok; Olver, Christine S; Bohn, Andrea A; Prenni, Jessica E

2014-06-01

Urine is an attractive biofluid for biomarker discovery as it is easy and minimally invasive to obtain. While numerous studies have focused on the characterization of human urine, much less research has focused on canine urine. The objectives of this study were to characterize the universal canine urinary proteome (both soluble and exosomal), to determine the overlap between the canine proteome and a representative human urinary proteome study, to generate a resource for future canine studies, and to determine the suitability of the dog as a large animal model for human diseases. The soluble and exosomal fractions of normal canine urine were characterized using liquid chromatography tandem mass spectrometry (LC-MS/MS). Biological Networks Gene Ontology (BiNGO) software was utilized to assign the canine urinary proteome to respective Gene Ontology categories, such as Cellular Component, Molecular Function, and Biological Process. Over 500 proteins were confidently identified in normal canine urine. Gene Ontology analysis revealed that exosomal proteins were largely derived from an intracellular location, while soluble proteins included both extracellular and membrane proteins. Exosome proteins were assigned to metabolic processes and localization, while soluble proteins were primarily annotated to specific localization processes. Several proteins identified in normal canine urine have previously been identified in human urine where these proteins are related to various extrarenal and renal diseases. The results of this study illustrate the potential of the dog as an animal model for human disease states and provide the framework for future studies of canine renal diseases. © 2014 American Society for Veterinary Clinical Pathology and European Society for Veterinary Clinical Pathology.

Single-cell proteomics: potential implications for cancer diagnostics.

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Gavasso, Sonia; Gullaksen, Stein-Erik; Skavland, Jørn; Gjertsen, Bjørn T

2016-01-01

Single-cell proteomics in cancer is evolving and promises to provide more accurate diagnoses based on detailed molecular features of cells within tumors. This review focuses on technologies that allow for collection of complex data from single cells, but also highlights methods that are adaptable to routine cancer diagnostics. Current diagnostics rely on histopathological analysis, complemented by mutational detection and clinical imaging. Though crucial, the information gained is often not directly transferable to defined therapeutic strategies, and predicting therapy response in a patient is difficult. In cancer, cellular states revealed through perturbed intracellular signaling pathways can identify functional mutations recurrent in cancer subsets. Single-cell proteomics remains to be validated in clinical trials where serial samples before and during treatment can reveal excessive clonal evolution and therapy failure; its use in clinical trials is anticipated to ignite a diagnostic revolution that will better align diagnostics with the current biological understanding of cancer.

Redefining the Breast Cancer Exosome Proteome by Tandem Mass Tag Quantitative Proteomics and Multivariate Cluster Analysis.

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Clark, David J; Fondrie, William E; Liao, Zhongping; Hanson, Phyllis I; Fulton, Amy; Mao, Li; Yang, Austin J

2015-10-20

Exosomes are microvesicles of endocytic origin constitutively released by multiple cell types into the extracellular environment. With evidence that exosomes can be detected in the blood of patients with various malignancies, the development of a platform that uses exosomes as a diagnostic tool has been proposed. However, it has been difficult to truly define the exosome proteome due to the challenge of discerning contaminant proteins that may be identified via mass spectrometry using various exosome enrichment strategies. To better define the exosome proteome in breast cancer, we incorporated a combination of Tandem-Mass-Tag (TMT) quantitative proteomics approach and Support Vector Machine (SVM) cluster analysis of three conditioned media derived fractions corresponding to a 10 000g cellular debris pellet, a 100 000g crude exosome pellet, and an Optiprep enriched exosome pellet. The quantitative analysis identified 2 179 proteins in all three fractions, with known exosomal cargo proteins displaying at least a 2-fold enrichment in the exosome fraction based on the TMT protein ratios. Employing SVM cluster analysis allowed for the classification 251 proteins as "true" exosomal cargo proteins. This study provides a robust and vigorous framework for the future development of using exosomes as a potential multiprotein marker phenotyping tool that could be useful in breast cancer diagnosis and monitoring disease progression.

Proteome Analysis of Human Arterial Tissue Discloses Associations Between the Vascular Content of Small Leucine-Rich Repeat Proteoglycans and Pulse Wave Velocity

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Lyck Hansen, Maria; Beck, Hans Christian; Irmukhamedov, Akhmadjon

2015-01-01

OBJECTIVES: We hypothesized that arterial stiffness is associated with changes in the arterial protein profile, particularly of extracellular matrix components. We aimed at determining differentially expressed proteins by quantitative proteome analysis in arterial tissue from patients with differ......OBJECTIVES: We hypothesized that arterial stiffness is associated with changes in the arterial protein profile, particularly of extracellular matrix components. We aimed at determining differentially expressed proteins by quantitative proteome analysis in arterial tissue from patients...... with different degrees of arterial stiffness. APPROACH AND RESULTS: Arterial stiffness, assessed by carotid-femoral pulse wave velocity (PWV), central blood pressure and augmentation index by pulse wave analysis were measured the day before surgery in a group of patients undergoing coronary artery bypass...... grafting. Protein extracts of well-defined, homogenous, nonatherosclerotic individual samples of the left mammary artery from 10 of these patients with high PWV and 9 with low PWV were compared by quantitative proteome analysis, using tandem mass tag labeling and nano-liquid chromatography mass...

Proteomic analysis of the secretory response of Aspergillus niger to D-maltose and D-xylose.

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José Miguel P Ferreira de Oliveira

Full Text Available Fungi utilize polysaccharide substrates through extracellular digestion catalyzed by secreted enzymes. Thus far, protein secretion by the filamentous fungus Aspergillus niger has mainly been studied at the level of individual proteins and by genome and transcriptome analyses. To extend these studies, a complementary proteomics approach was applied with the aim to investigate the changes in secretome and microsomal protein composition resulting from a shift to a high level secretion condition. During growth of A. niger on D-sorbitol, small amounts of D-maltose or D-xylose were used as inducers of the extracellular amylolytic and xylanolytic enzymes. Upon induction, protein compositions in the extracellular broth as well as in enriched secretory organelle (microsomal fractions were analyzed using a shotgun proteomics approach. In total 102 secreted proteins and 1,126 microsomal proteins were identified in this study. Induction by D-maltose or D-xylose resulted in the increase in specific extracellular enzymes, such as glucoamylase A on D-maltose and β-xylosidase D on D-xylose, as well as of microsomal proteins. This reflects the differential expression of selected genes coding for dedicated extracellular enzymes. As expected, the addition of extra D-sorbitol had no effect on the expression of carbohydrate-active enzymes, compared to addition of D-xylose or D-maltose. Furthermore, D-maltose induction caused an increase in microsomal proteins related to translation (e.g., Rpl15 and vesicular transport (e.g., the endosomal-cargo receptor Erv14. Millimolar amounts of the inducers D-maltose and D-xylose are sufficient to cause a direct response in specific protein expression levels. Also, after induction by D-maltose or D-xylose, the induced enzymes were found in microsomes and extracellular. In agreement with our previous findings for D-xylose induction, D-maltose induction leads to recruitment of proteins involved in proteasome-mediated degradation.

Proteomic analysis of the secretory response of Aspergillus niger to D-maltose and D-xylose.

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de Oliveira, José Miguel P Ferreira; van Passel, Mark W J; Schaap, Peter J; de Graaff, Leo H

2011-01-01

Fungi utilize polysaccharide substrates through extracellular digestion catalyzed by secreted enzymes. Thus far, protein secretion by the filamentous fungus Aspergillus niger has mainly been studied at the level of individual proteins and by genome and transcriptome analyses. To extend these studies, a complementary proteomics approach was applied with the aim to investigate the changes in secretome and microsomal protein composition resulting from a shift to a high level secretion condition. During growth of A. niger on D-sorbitol, small amounts of D-maltose or D-xylose were used as inducers of the extracellular amylolytic and xylanolytic enzymes. Upon induction, protein compositions in the extracellular broth as well as in enriched secretory organelle (microsomal) fractions were analyzed using a shotgun proteomics approach. In total 102 secreted proteins and 1,126 microsomal proteins were identified in this study. Induction by D-maltose or D-xylose resulted in the increase in specific extracellular enzymes, such as glucoamylase A on D-maltose and β-xylosidase D on D-xylose, as well as of microsomal proteins. This reflects the differential expression of selected genes coding for dedicated extracellular enzymes. As expected, the addition of extra D-sorbitol had no effect on the expression of carbohydrate-active enzymes, compared to addition of D-xylose or D-maltose. Furthermore, D-maltose induction caused an increase in microsomal proteins related to translation (e.g., Rpl15) and vesicular transport (e.g., the endosomal-cargo receptor Erv14). Millimolar amounts of the inducers D-maltose and D-xylose are sufficient to cause a direct response in specific protein expression levels. Also, after induction by D-maltose or D-xylose, the induced enzymes were found in microsomes and extracellular. In agreement with our previous findings for D-xylose induction, D-maltose induction leads to recruitment of proteins involved in proteasome-mediated degradation.

Characterization of rat primary trigeminal satellite glial cells and associated extracellular vesicles under normal and inflammatory conditions

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Vinterhøj, Hye Sook Han; Stensballe, Allan; Duroux, Meg

2018-01-01

Satellite glial cells (SGCs) in sensory ganglia contribute to the pathogenesis of chronic pain, potentially through mediating extracellular or paracrine signaling. Recently, extracellular vesicles (EVs) in the form of exosomes have been found to play an important role in cell-cell communication....... Results demonstrated that SGCs shed vesicles in the size range of exosomes (>150 nm) but with altered protein expression upon LPS-activation. Proteomic profiling of SGCs-shed EVs showed that a number of proteins were differentially regulated upon LPS stimulation such as junction plakoglobin and myosin 9...

Isolation and characterization of urinary extracellular vesicles: implications for biomarker discovery

NARCIS (Netherlands)

Merchant, M.L.; Rood, I.M.; Deegens, J.K.J.; Klein, J.B.

2017-01-01

Urine is a valuable diagnostic medium and, with the discovery of urinary extracellular vesicles, is viewed as a dynamic bioactive fluid. Extracellular vesicles are lipid-enclosed structures that can be classified into three categories: exosomes, microvesicles (or ectosomes) and apoptotic bodies.

Extracellular peptidases of the cereal pathogen Fusarium graminearum.

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Rohan George Thomas Lowe

2015-11-01

Full Text Available The plant pathogenic fungus Fusarium graminearum (Fgr creates economic and health risks in cereals agriculture. Fgr causes head blight (or scab of wheat and stalk rot of corn, reducing yield, degrading grain quality and polluting downstream food products with mycotoxins. Fungal plant pathogens must secrete proteases to access nutrition and to breakdown the structural protein component of the plant cell wall. Research into the proteolytic activity of Fgr is hindered by the complex nature of the suite of proteases secreted. We used a systems biology approach comprising genome analysis, transcriptomics and label-free quantitative proteomics to characterise the peptidases deployed by Fgr during growth. A combined analysis of published microarray transcriptome datasets revealed seven transcriptional groupings of peptidases based on in vitro growth, in planta growth, and sporulation behaviours. An orbitrap MS/MS proteomics technique defined the extracellular proteases secreted by Fusarium graminearum. A meta-classification based on sequence characters and transcriptional/translational activity in planta and in vitro provides a platform to develop control strategies that target Fgr peptidases.

Raman spectroscopy of single extracellular vesicles reveals subpopulations with varying membrane content (Conference Presentation)

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Smith, Zachary J.; Lee, Changwon; Rojalin, Tatu; Carney, Randy P.; Hazari, Sidhartha; Knudson, Alisha; Lam, Kit S.; Saari, Heikki; Lazaro Ibañez, Elisa; Viitala, Tapani; Laaksonen, Timo; Yliperttula, Marjo; Wachsmann-Hogiu, Sebastian

2016-03-01

Exosomes are small (~100nm) membrane bound vesicles excreted by cells as part of their normal biological processes. These extracellular vesicles are currently an area of intense research, since they were recently found to carry functional mRNA that allows transfer of proteins and other cellular instructions between cells. Exosomes have been implicated in a wide range of diseases, including cancer. Cancer cells are known to have increased exosome production, and may use those exosomes to prepare remote environments for metastasis. Therefore, there is a strong need to develop characterization methods to help understand the structure and function of these vesicles. However, current techniques, such as proteomics and genomics technologies, rely on aggregating a large amount of exosome material and reporting on chemical content that is averaged over many millions of exosomes. Here we report on the use of laser-tweezers Raman spectroscopy (LTRS) to probe individual vesicles, discovering distinct heterogeneity among exosomes both within a cell line, as well as between different cell lines. Through principal components analysis followed by hierarchical clustering, we have identified four "subpopulations" of exosomes shared across seven cell lines. The key chemical differences between these subpopulations, as determined by spectral analysis of the principal component loadings, are primarily related to membrane composition. Specifically, the differences can be ascribed to cholesterol content, cholesterol to phospholipid ratio, and surface protein expression. Thus, we have shown LTRS to be a powerful method to probe the chemical content of single extracellular vesicles.

Toxicoproteomics: serum proteomic pattern diagnostics for early detection of drug induced cardiac toxicities and cardioprotection.

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Petricoin, Emanuel F; Rajapaske, Vinodh; Herman, Eugene H; Arekani, Ali M; Ross, Sally; Johann, Donald; Knapton, Alan; Zhang, J; Hitt, Ben A; Conrads, Thomas P; Veenstra, Timothy D; Liotta, Lance A; Sistare, Frank D

2004-01-01

Proteomics is more than just generating lists of proteins that increase or decrease in expression as a cause or consequence of pathology. The goal should be to characterize the information flow through the intercellular protein circuitry which communicates with the extracellular microenvironment and then ultimately to the serum/plasma macroenvironment. The nature of this information can be a cause, or a consequence, of disease and toxicity based processes as cascades of reinforcing information percolate through the system and become reflected in changing proteomic information content of the circulation. Serum Proteomic Pattern Diagnostics is a new type of proteomic platform in which patterns of proteomic signatures from high dimensional mass spectrometry data are used as a diagnostic classifier. While this approach has shown tremendous promise in early detection of cancers, detection of drug-induced toxicity may also be possible with this same technology. Analysis of serum from rat models of anthracycline and anthracenedione induced cardiotoxicity indicate the potential clinical utility of diagnostic proteomic patterns where low molecular weight peptides and protein fragments may have higher accuracy than traditional biomarkers of cardiotoxicity such as troponins. These fragments may one day be harvested by circulating nanoparticles designed to absorb, enrich and amplify the diagnostic biomarker repertoire generated even at the critical initial stages of toxicity.

Cytoplasmic- and extracellular-proteome analysis of Diplodia seriata: a phytopathogenic fungus involved in grapevine decline

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Cobos Rebeca

2010-09-01

Full Text Available Abstract Background The phytopathogenic fungus Diplodia seriata, whose genome remains unsequenced, produces severe infections in fruit trees (fruit blight and grapevines. In this crop is recognized as one of the most prominent pathogens involved in grapevine trunk disease (or grapevine decline. This pathology can result in the death of adult plants and therefore it produces severe economical losses all around the world. To date no genes or proteins have been characterized in D. seriata that are involved in the pathogenicity process. In an effort to help identify potential gene products associated with pathogenicity and to gain a better understanding of the biology of D. seriata, we initiated a proteome-level study of the fungal mycelia and secretome. Results Intracellular and secreted proteins from D. seriata collected from liquid cultures were separated using two-dimensional gel electrophoresis. About 550 cytoplasmic proteins were reproducibly present in 3 independent extractions, being 53 identified by peptide mass fingerprinting and tandem mass spectrometry. The secretome analysis showed 75 secreted proteins reproducibly present in 3 biological replicates, being 16 identified. Several of the proteins had been previously identified as virulence factors in other fungal strains, although their contribution to pathogenicity in D. seriata remained to be analyzed. When D. seriata was grown in a medium supplemented with carboxymethylcellulose, 3 proteins were up-regulated and 30 down-regulated. Within the up-regulated proteins, two were identified as alcohol dehydrogenase and mitochondrial peroxyrredoxin-1, suggesting that they could play a significant role in the pathogenicity process. As for the 30 down-regulated proteins, 9 were identified being several of them involved in carbohydrate metabolism. Conclusions This study is the first report on proteomics on D. seriata. The proteomic data obtained will be important to understand the pathogenicity

Extracellular vesicles from parasitic helminths and their potential utility as vaccines.

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Mekonnen, Gebeyaw Getnet; Pearson, Mark; Loukas, Alex; Sotillo, Javier

2018-03-01

Helminths are multicellular parasites affecting nearly three billion people worldwide. To orchestrate a parasitic existence, helminths secrete different molecules, either in soluble form or contained within extracellular vesicles (EVs). EVs are secreted by most cell types and organisms, and have varied roles in intercellular communication, including immune modulation and pathogenesis. Areas covered: In this review, we describe the nucleic acid and proteomic composition of EVs from helminths, with a focus on the protein vaccine candidates present on the EV surface membrane, and discuss the potential utility of helminth EVs and their constituent proteins in the fight against helminth infections. Expert commentary: A significant number of proteins present in helminth-secreted EVs are known vaccine candidates. The characterization of helminth EV proteomes will shed light on host-pathogen interactions, facilitate the discovery of new diagnostic biomarkers, and provide a novel approach for the development of new control measures against helminth infections.

Dissecting plasmodesmata molecular composition by mass spectrometry-based proteomics.

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Emmanuelle Maria Françoise Bayer

2013-01-01

Full Text Available In plants, the intercellular communication through the membranous channels called plasmodesmata (PD; singular plasmodesma plays pivotal roles in the orchestration of development, defence responses and viral propagation. PD are dynamic structures embedded in the plant cell wall that are defined by specialised domains of the endoplasmic reticulum and the plasma membrane. PD structure and unique functions are guaranteed by their particular molecular composition. Yet, up to recent years and despite numerous approaches such as mutant screens, immunolocalisation or screening of random cDNAs, only few PD proteins had been conclusively identified and characterised. A clear breakthrough in the search of PD constituents came from mass-spectrometry-based proteomic approaches coupled with subcellular fractionation strategies. Due to their position, firmly anchored in the extracellular matrix, PD are notoriously difficult to isolate for biochemical analysis. Proteomic-based approaches have therefore first relied on the use of cell wall fractions containing embedded PD then on free PD fractions whereby PD membranes were released from the walls by enzymatic degradation. To discriminate between likely contaminants and PD protein candidates, bioinformatics tools have often been used in combination with proteomic approaches. GFP fusion proteins of selected candidates have confirmed the PD association of several protein families. Here we review the accomplishments and limitations of the proteomic based strategies to unravel the functional and structural complexity of PD. We also discuss the role of the identified PD associated proteins.

Dissecting plasmodesmata molecular composition by mass spectrometry-based proteomics.

Science.gov (United States)

Salmon, Magali S; Bayer, Emmanuelle M F

2012-01-01

In plants, the intercellular communication through the membranous channels called plasmodesmata (PD; singular plasmodesma) plays pivotal roles in the orchestration of development, defence responses, and viral propagation. PD are dynamic structures embedded in the plant cell wall that are defined by specialized domains of the endoplasmic reticulum (ER) and the plasma membrane (PM). PD structure and unique functions are guaranteed by their particular molecular composition. Yet, up to recent years and despite numerous approaches such as mutant screens, immunolocalization, or screening of random cDNAs, only few PD proteins had been conclusively identified and characterized. A clear breakthrough in the search of PD constituents came from mass-spectrometry-based proteomic approaches coupled with subcellular fractionation strategies. Due to their position, firmly anchored in the extracellular matrix, PD are notoriously difficult to isolate for biochemical analysis. Proteomic-based approaches have therefore first relied on the use of cell wall fractions containing embedded PD then on "free" PD fractions whereby PD membranes were released from the walls by enzymatic degradation. To discriminate between likely contaminants and PD protein candidates, bioinformatics tools have often been used in combination with proteomic approaches. GFP fusion proteins of selected candidates have confirmed the PD association of several protein families. Here we review the accomplishments and limitations of the proteomic-based strategies to unravel the functional and structural complexity of PD. We also discuss the role of the identified PD-associated proteins.

Lipid raft proteome reveals that oxidative phosphorylation system is associated with the plasma membrane.

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Kim, Bong-Woo; Lee, Chang Seok; Yi, Jae-Sung; Lee, Joo-Hyung; Lee, Joong-Won; Choo, Hyo-Jung; Jung, Soon-Young; Kim, Min-Sik; Lee, Sang-Won; Lee, Myung-Shik; Yoon, Gyesoon; Ko, Young-Gyu

2010-12-01

Although accumulating proteomic analyses have supported the fact that mitochondrial oxidative phosphorylation (OXPHOS) complexes are localized in lipid rafts, which mediate cell signaling, immune response and host-pathogen interactions, there has been no in-depth study of the physiological functions of lipid-raft OXPHOS complexes. Here, we show that many subunits of OXPHOS complexes were identified from the lipid rafts of human adipocytes, C2C12 myotubes, Jurkat cells and surface biotin-labeled Jurkat cells via shotgun proteomic analysis. We discuss the findings of OXPHOS complexes in lipid rafts, the role of the surface ATP synthase complex as a receptor for various ligands and extracellular superoxide generation by plasma membrane oxidative phosphorylation complexes.

High-resolution proteome maps of Bacillus licheniformis cells growing in minimal medium.

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Voigt, Birgit; Albrecht, Dirk; Sievers, Susanne; Becher, Dörte; Bongaerts, Johannes; Evers, Stefan; Schweder, Thomas; Maurer, Karl-Heinz; Hecker, Michael

2015-08-01

Bacillus licheniformis is an important host for the industrial production of enzymes mainly because of its ability to secrete large amounts of protein. We analyzed the proteome of B. licheniformis cells growing in a minimal medium. Beside the cytosolic proteome, the membrane and the extracellular proteome were studied. We could identify 1470 proteins; 1168 proteins were classified as cytosolic proteins, 195 proteins with membrane-spanning domains were classified as membrane proteins, and 107 proteins, with either putative signals peptides or flagellin-like sequences, were classified as secreted proteins. The identified proteins were grouped into functional categories and used to reconstruct cellular functions and metabolic pathways of growing B. licheniformis cells. The largest group was proteins with functions in basic metabolic pathways such as carbon metabolism, amino acid and nucleotide synthesis and synthesis of fatty acids and cofactors. Many proteins detected were involved in DNA replication, transcription, and translation. Furthermore, a high number of proteins employed in the transport of a wide variety of compounds were found to be expressed in the cells. All MS data have been deposited in the ProteomeXchange with identifier PXD000791 (http://proteomecentral.proteomexchange.org/dataset/PXD000791). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparative proteomic analysis of extracellular vesicles isolated by acoustic trapping or differential centrifugation

NARCIS (Netherlands)

Rezeli, Melinda; Gidlöf, Olof; Evander, Mikael; Bryl-Górecka, Paulina; Sathanoori, Ramasri; Gilje, Patrik; Pawlowski, Krzysztof; Horvatovich, Péter; Erlinge, David; Marko-Varga, György; Laurell, Thomas

2016-01-01

Extracellular vesicles (ECVs), including microparticles (MPs) and exosomes, are submicron membrane vesicles released by diverse cell types upon activation or stress. Circulating ECVs are potential reservoirs of disease biomarkers, and the complexity of these vesicles is significantly lower compared

Proteomic profiling of Mycobacterium tuberculosis identifies nutrient-starvation-responsive toxin-antitoxin systems

DEFF Research Database (Denmark)

Albrethsen, Jakob; Agner, Jeppe; Piersma, Sander R

2013-01-01

In order to successfully enter the latent stage, Mycobacterium tuberculosis must adapt to conditions such as nutrient limitation and hypoxia. In vitro models that mimic latent infection are valuable tools for describing the changes in metabolism that occur when the bacterium exists in a non......-growing form. We used two complementary proteomic approaches, label-free LC-MS/MS analysis and two-dimensional difference gel electrophoresis, to determine the proteome profile of extracellular proteins from M. tuberculosis cultured under nutrient starvation. Through the label-free LC-MS/MS analysis......, significant differences in the overall metabolism during nutrient starvation were detected. Notably, members of the toxin-antitoxin systems were present in larger quantities in nutrient-starved cultures, supporting a role for these global modules as M. tuberculosis switches its metabolism into dormancy...

High-resolution proteomic and lipidomic analysis of exosomes and microvesicles from different cell sources

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Reka A. Haraszti

2016-11-01

Full Text Available Extracellular vesicles (EVs, including exosomes and microvesicles (MVs, are explored for use in diagnostics, therapeutics and drug delivery. However, little is known about the relationship of protein and lipid composition of EVs and their source cells. Here, we report high-resolution lipidomic and proteomic analyses of exosomes and MVs derived by differential ultracentrifugation from 3 different cell types: U87 glioblastoma cells, Huh7 hepatocellular carcinoma cells and human bone marrow-derived mesenchymal stem cells (MSCs. We identified 3,532 proteins and 1,961 lipid species in the screen. Exosomes differed from MVs in several different areas: (a The protein patterns of exosomes were more likely different from their cells of origin than were the protein patterns of MVs; (b The proteomes of U87 and Huh7 exosomes were similar to each other but different from the proteomes of MSC exosomes, whereas the lipidomes of Huh7 and MSC exosomes were similar to each other but different from the lipidomes of U87 exosomes; (c exosomes exhibited proteins of extracellular matrix, heparin-binding, receptors, immune response and cell adhesion functions, whereas MVs were enriched in endoplasmic reticulum, proteasome and mitochondrial proteins. Exosomes and MVs also differed in their types of lipid contents. Enrichment in glycolipids and free fatty acids characterized exosomes, whereas enrichment in ceramides and sphingomyelins characterized MVs. Furthermore, Huh7 and MSC exosomes were specifically enriched in cardiolipins; U87 exosomes were enriched in sphingomyelins. This study comprehensively analyses the protein and lipid composition of exosomes, MVs and source cells in 3 different cell types.

Enhanced extracellular chitinase production in Pseudomonas fluorescens: biotechnological implications

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Azhar Alhasawi

2017-06-01

Full Text Available Chitin is an important renewable biomass of immense commercial interest. The processing of this biopolymer into value-added products in an environmentally-friendly manner necessitates its conversion into N-acetyl glucosamine (NAG, a reaction mediated by the enzyme chitinase. Here we report on the ability of the soil microbe Pseudomonas fluorescens to secrete copious amounts of chitinase in the spent fluid when cultured in mineral medium with chitin as the sole source of carbon and nitrogen. Although chitinase was detected in various cellular fractions, the enzyme was predominantly localized in the extracellular component that was also rich in NAG and glucosamine. Maximal amounts of chitinase with a specific activity of 80 µmol NAG produced mg–1 protein min–1 was obtained at pH 8 after 6 days of growth in medium with 0.5 g of chitin. In-gel activity assays and Western blot studies revealed three isoenzymes. The enzyme had an optimal activity at pH 10 and a temperature range of 22–38 ℃. It was stable for up to 3 months. Although it showed optimal specificity toward chitin, the enzyme did readily degrade shrimp shells. When these shells (0.1 g were treated with the extracellular chitinase preparation, NAG [3 mmoles (0.003 g-mol] was generated in 6 h. The extracellular nature of the enzyme coupled with its physico-chemical properties make this chitinase an excellent candidate for biotechnological applications.

Proteomics Core

Data.gov (United States)

Federal Laboratory Consortium — Proteomics Core is the central resource for mass spectrometry based proteomics within the NHLBI. The Core staff help collaborators design proteomics experiments in a...

SILK FIBRE DEGRADATION AND ANALYSIS BY PROTEOMICS

Directory of Open Access Journals (Sweden)

YUKSELOGLU S.Muge

2016-05-01

Full Text Available Silk is one of the promising natural fibres and has a long established history in textile production throughout the centuries. Silk is produced by cultured silk worms, spiders, scorpions, mites and flies. It is extracellular proteinaceous fibres which consist of highly crystalline and insoluble proteins, the fibroins glued with sericin and an amourphous protein. On the other hand, understanding and controlling the degradation of protein materials are important for determining quality and the value of appearance retention in textiles. Hence, for silk textiles, appearance retention is critical value for the quality. And this is one of the key properties directly related to the degree and nature of protein degradation. It is therefore necessary to understand the silk composition and damage to obtain good conservation treatments and long-term preservation especially for the historical silk fabrics. In this study, silk fibre and its properties are briefly introduced along with images on their fibre damages. Additionally, proteomics method which helps to understand the degradation at the molecular level in textiles is introduced. Finally, proteomic evaluation of silk is summarized according to the researchers carried out in the literature.

Power of Proteomics in Linking Oxidative Stress and Female Infertility

Science.gov (United States)

Gupta, Sajal; Sharma, Rakesh; Agarwal, Ashok

2014-01-01

Endometriosis, PCOS, and unexplained infertility are currently the most common diseases rendering large numbers of women infertile worldwide. Oxidative stress, due to its deleterious effects on proteins and nucleic acids, is postulated to be the one of the important mechanistic pathways in differential expression of proteins and in these diseases. The emerging field of proteomics has allowed identification of proteins involved in cell cycle, as antioxidants, extracellular matrix (ECM), cytoskeleton, and their linkage to oxidative stress in female infertility related diseases. The aim of this paper is to assess the association of oxidative stress and protein expression in the reproductive microenvironments such as endometrial fluid, peritoneal fluid, and follicular fluid, as well as reproductive tissues and serum. The review also highlights the literature that proposes the use of the fertility related proteins as potential biomarkers for noninvasive and early diagnosis of the aforementioned diseases rather than utilizing the more invasive methods used currently. The review will highlight the power of proteomic profiles identified in infertility related disease conditions and their linkage with underlying oxidative stress. The power of proteomics will be reviewed with regard to eliciting molecular mechanisms for early detection and management of these infertility related conditions. PMID:24900998

Power of Proteomics in Linking Oxidative Stress and Female Infertility

Directory of Open Access Journals (Sweden)

Sajal Gupta

2014-01-01

Full Text Available Endometriosis, PCOS, and unexplained infertility are currently the most common diseases rendering large numbers of women infertile worldwide. Oxidative stress, due to its deleterious effects on proteins and nucleic acids, is postulated to be the one of the important mechanistic pathways in differential expression of proteins and in these diseases. The emerging field of proteomics has allowed identification of proteins involved in cell cycle, as antioxidants, extracellular matrix (ECM, cytoskeleton, and their linkage to oxidative stress in female infertility related diseases. The aim of this paper is to assess the association of oxidative stress and protein expression in the reproductive microenvironments such as endometrial fluid, peritoneal fluid, and follicular fluid, as well as reproductive tissues and serum. The review also highlights the literature that proposes the use of the fertility related proteins as potential biomarkers for noninvasive and early diagnosis of the aforementioned diseases rather than utilizing the more invasive methods used currently. The review will highlight the power of proteomic profiles identified in infertility related disease conditions and their linkage with underlying oxidative stress. The power of proteomics will be reviewed with regard to eliciting molecular mechanisms for early detection and management of these infertility related conditions.

Proteomic analysis of Aspergillus niger type strain (MUM 03.01) after accelerated freeze-drying preservation

OpenAIRE

Simões, Marta Filipa Jesus Freitas; Santos, Cledir; Rampitsch, Christof; Lima, Nelson

2013-01-01

Industrial processes based on biotechnology require a reliable source of microbial cultures, raising a need for the microbial safe long-term storage. The expanding biotechnological applications of Aspergillus niger requires effective long-term preservation that maintains their viability and also their physiological and genetic features [I]. From the best of our knowledge, proteomic analysis for monitoring the effect of preservation techniques on fungai intra- and extracellular ...

Comparative proteomic exploration of whey proteins in human and bovine colostrum and mature milk using iTRAQ-coupled LC-MS/MS.

Science.gov (United States)

Yang, Mei; Cao, Xueyan; Wu, Rina; Liu, Biao; Ye, Wenhui; Yue, Xiqing; Wu, Junrui

2017-09-01

Whey, an essential source of dietary nutrients, is widely used in dairy foods for infants. A total of 584 whey proteins in human and bovine colostrum and mature milk were identified and quantified by the isobaric tag for relative and absolute quantification (iTRAQ) proteomic method. The 424 differentially expressed whey proteins were identified and analyzed according to gene ontology (GO) annotation, Kyoto encyclopedia of genes and genomes (KEGG) pathway, and multivariate statistical analysis. Biological processes principally involved biological regulation and response to stimulus. Major cellular components were extracellular region part and extracellular space. The most prevalent molecular function was protein binding. Twenty immune-related proteins and 13 proteins related to enzyme regulatory activity were differentially expressed in human and bovine milk. Differentially expressed whey proteins participated in many KEGG pathways, including major complement and coagulation cascades and in phagosomes. Whey proteins show obvious differences in expression in human and bovine colostrum and mature milk, with consequences for biological function. The results here increase our understanding of different whey proteomes, which could provide useful information for the development and manufacture of dairy products and nutrient food for infants. The advanced iTRAQ proteomic approach was used to analyze differentially expressed whey proteins in human and bovine colostrum and mature milk.

The proteome of neurofilament-containing protein aggregates in blood

Directory of Open Access Journals (Sweden)

Rocco Adiutori

2018-07-01

Full Text Available Protein aggregation in biofluids is a poorly understood phenomenon. Under normal physiological conditions, fluid-borne aggregates may contain plasma or cell proteins prone to aggregation. Recent observations suggest that neurofilaments (Nf, the building blocks of neurons and a biomarker of neurodegeneration, are included in high molecular weight complexes in circulation. The composition of these Nf-containing hetero-aggregates (NCH may change in systemic or organ-specific pathologies, providing the basis to develop novel disease biomarkers. We have tested ultracentrifugation (UC and a commercially available protein aggregate binder, Seprion PAD-Beads (SEP, for the enrichment of NCH from plasma of healthy individuals, and then characterised the Nf content of the aggregate fractions using gel electrophoresis and their proteome by mass spectrometry (MS. Western blot analysis of fractions obtained by UC showed that among Nf isoforms, neurofilament heavy chain (NfH was found within SDS-stable high molecular weight aggregates. Shotgun proteomics of aggregates obtained with both extraction techniques identified mostly cell structural and to a lesser extent extra-cellular matrix proteins, while functional analysis revealed pathways involved in inflammatory response, phagosome and prion-like protein behaviour. UC aggregates were specifically enriched with proteins involved in endocrine, metabolic and cell-signalling regulation. We describe the proteome of neurofilament-containing aggregates isolated from healthy individuals biofluids using different extraction methods.

Comparative proteomic assessment of matrisome enrichment methodologies

Science.gov (United States)

Krasny, Lukas; Paul, Angela; Wai, Patty; Howard, Beatrice A.; Natrajan, Rachael C.; Huang, Paul H.

2016-01-01

The matrisome is a complex and heterogeneous collection of extracellular matrix (ECM) and ECM-associated proteins that play important roles in tissue development and homeostasis. While several strategies for matrisome enrichment have been developed, it is currently unknown how the performance of these different methodologies compares in the proteomic identification of matrisome components across multiple tissue types. In the present study, we perform a comparative proteomic assessment of two widely used decellularisation protocols and two extraction methods to characterise the matrisome in four murine organs (heart, mammary gland, lung and liver). We undertook a systematic evaluation of the performance of the individual methods on protein yield, matrisome enrichment capability and the ability to isolate core matrisome and matrisome-associated components. Our data find that sodium dodecyl sulphate (SDS) decellularisation leads to the highest matrisome enrichment efficiency, while the extraction protocol that comprises chemical and trypsin digestion of the ECM fraction consistently identifies the highest number of matrisomal proteins across all types of tissue examined. Matrisome enrichment had a clear benefit over non-enriched tissue for the comprehensive identification of matrisomal components in murine liver and heart. Strikingly, we find that all four matrisome enrichment methods led to significant losses in the soluble matrisome-associated proteins across all organs. Our findings highlight the multiple factors (including tissue type, matrisome class of interest and desired enrichment purity) that influence the choice of enrichment methodology, and we anticipate that these data will serve as a useful guide for the design of future proteomic studies of the matrisome. PMID:27589945

Neutrophil extracellular traps in patients with pulmonary tuberculosis

NARCIS (Netherlands)

van der Meer, Anne Jan; Zeerleder, Sacha; Blok, Dana C.; Kager, Liesbeth M.; Lede, Ivar O.; Rahman, Wahid; Afroz, Rumana; Ghose, Aniruddha; Visser, Caroline E.; Zahed, Abu Shahed Md; Husain, Md Anwar; Alam, Khan Mashrequl; Barua, Pravat Chandra; Hassan, Mahtabuddin; Tayab, Md Abu; Dondorp, Arjen M.; van der Poll, Tom

2017-01-01

Tuberculosis is a devastating infectious disease causing many deaths worldwide. Recent investigations have implicated neutrophil extracellular traps (NETs) in the host response to tuberculosis. The aim of the current study was to obtain evidence for NETs release in the circulation during human

Extracellular clusterin promotes neuronal network complexity in vitro

DEFF Research Database (Denmark)

Wicher, Grzegorz; Velsecchi, Isabel; Charnay, Yves

2008-01-01

Clusterin (apolipoprotein J), a highly conserved amphiphatic glycoprotein and chaperone, has been implicated in a wide range of physiological and pathological processes. As a secreted protein, clusterin has been shown to act extracellularly where it is involved in lipid transportation and clearan...

Clinical proteomics

DEFF Research Database (Denmark)

Albrethsen, Jakob; Frederiksen, Hanne; Johannsen, Trine Holm

2018-01-01

Clinical proteomics aims to deliver cost-effective multiplexing of potentially hundreds of diagnostic proteins, including distinct protein isoforms. The analytical strategy known as targeted proteomics is particularly promising because it is compatible with robust mass spectrometry (MS)-platforms...... standards and calibrants. The present challenge is to examine if targeted proteomics of IGF-I can truly measure up to the routine performance that must be expected from a clinical testing platform.......Clinical proteomics aims to deliver cost-effective multiplexing of potentially hundreds of diagnostic proteins, including distinct protein isoforms. The analytical strategy known as targeted proteomics is particularly promising because it is compatible with robust mass spectrometry (MS......)-platforms already implemented in many clinical laboratories for routine quantitation of small molecules (i.e. uHPLC coupled to triple-quadrupole MS). Progress in targeted proteomics of circulating insulin-like growth factor 1 (IGF-I) have provided valuable insights about tryptic peptides, transitions, internal...

Proteomic Analysis of the Secretory Response of Aspergillus niger to D-Maltose and D-Xylose

OpenAIRE

Ferreira de Oliveira, José Miguel P.; van Passel, Mark W. J.; Schaap, Peter J.; de Graaff, Leo H.

2011-01-01

Fungi utilize polysaccharide substrates through extracellular digestion catalyzed by secreted enzymes. Thus far, protein secretion by the filamentous fungus Aspergillus niger has mainly been studied at the level of individual proteins and by genome and transcriptome analyses. To extend these studies, a complementary proteomics approach was applied with the aim to investigate the changes in secretome and microsomal protein composition resulting from a shift to a high level secretion condition....

A catalogue of human secreted proteins and its implications

Directory of Open Access Journals (Sweden)

Shivakumar Keerthikumar

2016-11-01

Full Text Available Under both normal and pathological conditions, cells secrete variety of proteins through classical and non-classical secretory pathways into the extracellular space. Majority of these proteins represent pathophysiology of the cell from which it is secreted. Recently, though more than 92% of the protein coding genes has been mapped by human proteome map project, but number of those proteins that constitutes secretome of the cell still remains elusive. Secreted proteins or the secretome can be accessible in bodily fluids and hence are considered as potential biomarkers to discriminate between healthy and diseased individuals. In order to facilitate the biomarker discovery and to further aid clinicians and scientists working in these arenas, we have compiled and catalogued secreted proteins from the human proteome using integrated bioinformatics approach. In this study, nearly 14% of the human proteome is likely to be secreted through classical and non-classical secretory pathways. Out of which, ~38% of these secreted proteins were found in extracellular vesicles including exosomes and shedding microvesicles. Among these secreted proteins, 94% were detected in human bodily fluids including blood, plasma, serum, saliva, semen, tear and urine. We anticipate that this high confidence list of secreted proteins could serve as a compendium of candidate biomarkers. In addition, the catalogue may provide functional insights in understanding the molecular mechanisms involved in various physiological and pathophysiological conditions of the cell.

Population-specific plasma proteomes of marine and freshwater three-spined sticklebacks (Gasterosteus aculeatus).

Science.gov (United States)

Kültz, Dietmar; Li, Johnathon; Zhang, Xuezhen; Villarreal, Fernando; Pham, Tuan; Paguio, Darlene

2015-12-01

Molecular phenotypes that distinguish resident marine (Bodega Harbor) from landlocked freshwater (FW, Lake Solano) three-spined sticklebacks were revealed by label-free quantitative proteomics. Secreted plasma proteins involved in lipid transport, blood coagulation, proteolysis, plasminogen-activating cascades, extracellular stimulus responses, and immunity are most abundant in this species. Globulins and albumins are much less abundant than in mammalian plasma. Unbiased quantitative proteome profiling identified 45 highly population-specific plasma proteins. Population-specific abundance differences were validated by targeted proteomics based on data-independent acquisition. Gene ontology enrichment analyses and known functions of population-specific plasma proteins indicate enrichment of processes controlling cell adhesion, tissue remodeling, proteolytic processing, and defense signaling in marine sticklebacks. Moreover, fetuin B and leukocyte cell derived chemotaxin 2 are much more abundant in marine fish. These proteins promote bone morphogenesis and likely contribute to population-specific body armor differences. Plasma proteins enriched in FW fish promote translation, heme biosynthesis, and lipid transport, suggesting a greater presence of plasma microparticles. Many prominent population-specific plasma proteins (e.g. apoptosis-associated speck-like protein containing a CARD) lack any homolog of known function or adequate functional characterization. Their functional characterization and the identification of population-specific environmental contexts and selective pressures that cause plasma proteome diversification are future directions emerging from this study. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mass spectrometry-based proteomic exploration of the human immune system: focus on the inflammasome, global protein secretion, and T cells.

Science.gov (United States)

Nyman, Tuula A; Lorey, Martina B; Cypryk, Wojciech; Matikainen, Sampsa

2017-05-01

The immune system is our defense system against microbial infections and tissue injury, and understanding how it works in detail is essential for developing drugs for different diseases. Mass spectrometry-based proteomics can provide in-depth information on the molecular mechanisms involved in immune responses. Areas covered: Summarized are the key immunology findings obtained with MS-based proteomics in the past five years, with a focus on inflammasome activation, global protein secretion, mucosal immunology, immunopeptidome and T cells. Special focus is on extracellular vesicle-mediated protein secretion and its role in immune responses. Expert commentary: Proteomics is an essential part of modern omics-scale immunology research. To date, MS-based proteomics has been used in immunology to study protein expression levels, their subcellular localization, secretion, post-translational modifications, and interactions in immune cells upon activation by different stimuli. These studies have made major contributions to understanding the molecular mechanisms involved in innate and adaptive immune responses. New developments in proteomics offer constantly novel possibilities for exploring the immune system. Examples of these techniques include mass cytometry and different MS-based imaging approaches which can be widely used in immunology.

Proteomic analysis of Aspergillus fumigatus - clinical implications.

Science.gov (United States)

Moloney, Nicola M; Owens, Rebecca A; Doyle, Sean

2016-07-01

Aspergillus fumigatus is a ubiquitous saprophytic fungus capable of producing small airborne spores, which are frequently inhaled by humans. In healthy individuals, the fungus is rapidly cleared by innate mechanisms, including immune cells. However, in individuals with impaired lung function or immunosuppression the spores can germinate and prompt severe allergic responses, and disease with limited or extensive invasiveness. The traits that make A. fumigatus a successful colonizer and pathogen of humans are multi-factorial. Thus, a global investigative approach is required to elucidate the mechanisms utilized by the fungus to cause disease. Expert commentary: In doing so, a better understanding of disease pathology can be achieved with improved therapeutic/diagnostic solutions, thereby improving patient outcome. Proteomic analysis permits such investigations and recent work has yielded insight into these mechanisms.

The extracellular proteome of two Bifidobacterium species reveals different adaptation strategies to low iron conditions

OpenAIRE

Vazquez-Gutierrez, Pamela; Stevens, Marc J. A.; Gehrig, Peter; Barkow-Oesterreicher, Simon; Lacroix, Christophe; Chassard, Christophe

2017-01-01

Background Bifidobacteria are among the first anaerobic bacteria colonizing the gut. Bifidobacteria require iron for growth and their iron-sequestration mechanisms are important for their fitness and possibly inhibit enteropathogens. Here we used combined genomic and proteomic analyses to characterize adaptations to low iron conditions of B. kashiwanohense PV20-2 and B. pseudolongum PV8-2, 2 strains isolated from the feces of iron-deficient African infants and selected for their high iron-seq...

Detection and transmission of extracellular fac-tor producing Streptococcus suis serotype 2 strains in pigs

NARCIS (Netherlands)

Swildens, B.

2009-01-01

DETECTION AND TRANSMISSION OF EXTRACELLULAR FACTOR PRODUCING STREPTOCOCCUS SUIS SEROTYPE 2 STRAINS IN PIGS INTRODUCTION Streptococcus suis (S.suis) has been implicated in the etiology of many diseases among which meningitis in pigs. The virulent extracellular factor-positive strains of S.suis

Proteomic signatures implicate cAMP in light and temperature responses in Arabidopsis thaliana

KAUST Repository

Thomas, Ludivine

2013-05-01

The second messenger 3\\'-5\\'-cyclic adenosine monophosphate (cAMP) and adenylyl cyclases (ACs), enzymes that catalyse the formation of cAMP from ATP, are increasingly recognized as important signaling molecules in a number of physiological responses in higher plants. Here we used proteomics to identify cAMP-dependent protein signatures in Arabidopsis thaliana and identify a number of differentially expressed proteins with a role in light- and temperature-dependent responses, notably photosystem II subunit P-1, plasma membrane associated cation-binding protein and chaperonin 60 β. Based on these proteomics results we conclude that, much like in cyanobacteria, algae and fungi, cAMP may have a role in light signaling and the regulation of photosynthesis as well as responses to temperature and we speculate that ACs could act as light and/or temperature sensors in higher plants. Biological significance: This current study is significant since it presents the first proteomic response to cAMP, a novel and key second messenger in plants. It will be relevant to researchers in plant physiology and in particular those with an interest in second messengers and their role in biotic and abiotic stress responses. © 2013 Elsevier B.V.

Global analysis of the yeast osmotic stress response by quantitative proteomics

DEFF Research Database (Denmark)

Soufi, Boumediene; Kelstrup, C.D.; Stoehr, G.

2009-01-01

a comprehensive, quantitative, and time-resolved analysis using high-resolution mass spectrometry of phospho-proteome and proteome changes in response to osmotic stress in yeast. We identified 5534 unique phosphopeptide variants and 3383 yeast proteins. More than 15% of the detected phosphorylation site status...... changed more than two-fold within 5 minutes of treatment. Many of the corresponding phosphoproteins are involved in the early response to environmental stress. Surprisingly, we find that 158 regulated phosphorylation sites are potential substrates of basophilic kinases as opposed to the classical proline......-directed MAP kinase network implicated in stress response mechanisms such as p38 and HOG pathways. Proteome changes reveal an increase in abundance of more than one hundred proteins after 20 min of salt stress. Many of these are involved in the cellular response to increased osmolarity, which include proteins...

Characterization of the porcine synovial fluid proteome and a comparison to the plasma proteome

Directory of Open Access Journals (Sweden)

Tue Bjerg Bennike

2015-12-01

In addition, we analyzed the proteome of human plasma, and compared the proteomes to the obtained porcine synovial fluid proteome. The proteome of the two body fluids were found highly similar, underlining the detected plasma derived nature of many synovial fluid components. The healthy porcine synovial fluid proteomics data, human rheumatoid arthritis synovial fluid proteomics data used in the method optimization, human plasma proteomics data, and search results, have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD000935.

ProteomicsDB.

Science.gov (United States)

Schmidt, Tobias; Samaras, Patroklos; Frejno, Martin; Gessulat, Siegfried; Barnert, Maximilian; Kienegger, Harald; Krcmar, Helmut; Schlegl, Judith; Ehrlich, Hans-Christian; Aiche, Stephan; Kuster, Bernhard; Wilhelm, Mathias

2018-01-04

ProteomicsDB (https://www.ProteomicsDB.org) is a protein-centric in-memory database for the exploration of large collections of quantitative mass spectrometry-based proteomics data. ProteomicsDB was first released in 2014 to enable the interactive exploration of the first draft of the human proteome. To date, it contains quantitative data from 78 projects totalling over 19k LC-MS/MS experiments. A standardized analysis pipeline enables comparisons between multiple datasets to facilitate the exploration of protein expression across hundreds of tissues, body fluids and cell lines. We recently extended the data model to enable the storage and integrated visualization of other quantitative omics data. This includes transcriptomics data from e.g. NCBI GEO, protein-protein interaction information from STRING, functional annotations from KEGG, drug-sensitivity/selectivity data from several public sources and reference mass spectra from the ProteomeTools project. The extended functionality transforms ProteomicsDB into a multi-purpose resource connecting quantification and meta-data for each protein. The rich user interface helps researchers to navigate all data sources in either a protein-centric or multi-protein-centric manner. Several options are available to download data manually, while our application programming interface enables accessing quantitative data systematically. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Using the Ubiquitin-modified Proteome to Monitor Distinct and Spatially Restricted Protein Homeostasis Dysfunction.

Science.gov (United States)

Gendron, Joshua M; Webb, Kristofor; Yang, Bing; Rising, Lisa; Zuzow, Nathan; Bennett, Eric J

2016-08-01

Protein homeostasis dysfunction has been implicated in the development and progression of aging related human pathologies. There is a need for the establishment of quantitative methods to evaluate global protein homoeostasis function. As the ubiquitin (ub) proteasome system plays a key role in regulating protein homeostasis, we applied quantitative proteomic methods to evaluate the sensitivity of site-specific ubiquitylation events as markers for protein homeostasis dysfunction. Here, we demonstrate that the ub-modified proteome can exceed the sensitivity of engineered fluorescent reporters as a marker for proteasome dysfunction and can provide unique signatures for distinct proteome challenges which is not possible with engineered reporters. We demonstrate that combining ub-proteomics with subcellular fractionation can effectively separate degradative and regulatory ubiquitylation events on distinct protein populations. Using a recently developed potent inhibitor of the critical protein homeostasis factor p97/VCP, we demonstrate that distinct insults to protein homeostasis function can elicit robust and largely unique alterations to the ub-modified proteome. Taken together, we demonstrate that proteomic approaches to monitor the ub-modified proteome can be used to evaluate global protein homeostasis and can be used to monitor distinct functional outcomes for spatially separated protein populations. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Building ProteomeTools based on a complete synthetic human proteome

Science.gov (United States)

Zolg, Daniel P.; Wilhelm, Mathias; Schnatbaum, Karsten; Zerweck, Johannes; Knaute, Tobias; Delanghe, Bernard; Bailey, Derek J.; Gessulat, Siegfried; Ehrlich, Hans-Christian; Weininger, Maximilian; Yu, Peng; Schlegl, Judith; Kramer, Karl; Schmidt, Tobias; Kusebauch, Ulrike; Deutsch, Eric W.; Aebersold, Ruedi; Moritz, Robert L.; Wenschuh, Holger; Moehring, Thomas; Aiche, Stephan; Huhmer, Andreas; Reimer, Ulf; Kuster, Bernhard

2018-01-01

The ProteomeTools project builds molecular and digital tools from the human proteome to facilitate biomedical and life science research. Here, we report the generation and multimodal LC-MS/MS analysis of >330,000 synthetic tryptic peptides representing essentially all canonical human gene products and exemplify the utility of this data. The resource will be extended to >1 million peptides and all data will be shared with the community via ProteomicsDB and proteomeXchange. PMID:28135259

Clinical veterinary proteomics: Techniques and approaches to decipher the animal plasma proteome.

Science.gov (United States)

Ghodasara, P; Sadowski, P; Satake, N; Kopp, S; Mills, P C

2017-12-01

Over the last two decades, technological advancements in the field of proteomics have advanced our understanding of the complex biological systems of living organisms. Techniques based on mass spectrometry (MS) have emerged as powerful tools to contextualise existing genomic information and to create quantitative protein profiles from plasma, tissues or cell lines of various species. Proteomic approaches have been used increasingly in veterinary science to investigate biological processes responsible for growth, reproduction and pathological events. However, the adoption of proteomic approaches by veterinary investigators lags behind that of researchers in the human medical field. Furthermore, in contrast to human proteomics studies, interpretation of veterinary proteomic data is difficult due to the limited protein databases available for many animal species. This review article examines the current use of advanced proteomics techniques for evaluation of animal health and welfare and covers the current status of clinical veterinary proteomics research, including successful protein identification and data interpretation studies. It includes a description of an emerging tool, sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS), available on selected mass spectrometry instruments. This newly developed data acquisition technique combines advantages of discovery and targeted proteomics approaches, and thus has the potential to advance the veterinary proteomics field by enhancing identification and reproducibility of proteomics data. Copyright © 2017 Elsevier Ltd. All rights reserved.

Trash or Treasure: extracellular microRNAs and cell-to-cell communication

Directory of Open Access Journals (Sweden)

Nobuyoshi eKosaka

2013-09-01

Full Text Available Circulating RNAs in human body fluids are promising candidates for diagnostic purposes. However, the biological significance of circulating RNAs remains elusive. Recently, small non-coding RNAs, microRNAs (miRNAs, were isolated from multiple human body fluids, and these circulating miRNAs have been implicated as novel disease biomarkers. Concurrently, miRNAs were also identified in the extracellular space associated with extracellular vesicles (EVs, which are small membrane vesicles secreted from various types of cells. The function of these secreted miRNAs has been revealed in several papers. Circulating miRNAs have been experimentally found to be associated with EVs, however, other types of extracellular miRNAs were also described. This review discusses studies related to extracellular miRNAs, including circulating miRNAs and secreted miRNAs, to highlight the importance of studying not only secreted miRNAs but also circulating miRNAs to determine the contribution of extracellular miRNAs especially in cancer development.

Composition of the Extracellular Matrix of Lymphatic Novel Threadlike Structures: Is It Keratin?

Directory of Open Access Journals (Sweden)

Hyub Huh

2013-01-01

Full Text Available Background. The lumen of novel threadlike structures (NTSs is enclosed by a single layer of endothelial cells surrounded by extracellular matrix (ECM. We hypothesized that collagen may be a component of the ECM associated with lymphatic NTSs. Methods. Six female New Zealand white rabbits were anesthetized, and the NTS structures within lymphatic vessels were identified by contrast-enhanced stereomicroscopy or alcian blue staining. Isolated NTS specimens were stained with acridine orange, YOYO-1, and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI. The structural and molecular composition of the ECM was investigated using transmission electron microscopy (TEM, electrospray ionization-mass spectrometry, and proteomic analysis. Results. The lymph vessel wall was stained red by DiI, and rod-shaped nuclei were stained green by YOYO-1. The area surrounding the NTS was also stained red and contained green rod-shaped nuclei. TEM images showed that the NTS consisted of many ECM fibers and the ECM fibers appeared to be ~100 nm in diameter and had narrowly spaced striated bands. Proteomic analysis of the lymphatic NTS-associated ECM identified 4 proteins: keratin 10, cytokeratin 3, cytokeratin 12, and soluble adenylyl cyclase. Conclusion. The TEM study suggested that the lymphatic NTS-associated ECM did not contain collagen. This was confirmed by proteomic analysis, which showed that keratin was the major component of the ECM.

Proteomic Profiling of the Pituitary Gland in Studies of Psychiatric Disorders.

Science.gov (United States)

Krishnamurthy, Divya; Rahmoune, Hassan; Guest, Paul C

2017-01-01

Psychiatric disorders have been associated with perturbations of the hypothalamic-pituitary-adrenal axis. Therefore, proteomic studies of the pituitary gland have the potential to provide new insights into the underlying pathways affected in these conditions as well as identify new biomarkers or targets for use in developing improved medications. This chapter describes a protocol for preparation of pituitary protein extracts followed by characterization of the pituitary proteome by label-free liquid chromatography-tandem mass spectrometry in expression mode (LC-MS E ). The main focus was on establishing a method for identifying the major pituitary hormones and accessory proteins as many of these have already been implicated in psychiatric diseases.

In Silico Functional Networks Identified in Fish Nucleated Red Blood Cells by Means of Transcriptomic and Proteomic Profiling.

Science.gov (United States)

Puente-Marin, Sara; Nombela, Iván; Ciordia, Sergio; Mena, María Carmen; Chico, Verónica; Coll, Julio; Ortega-Villaizan, María Del Mar

2018-04-09

Nucleated red blood cells (RBCs) of fish have, in the last decade, been implicated in several immune-related functions, such as antiviral response, phagocytosis or cytokine-mediated signaling. RNA-sequencing (RNA-seq) and label-free shotgun proteomic analyses were carried out for in silico functional pathway profiling of rainbow trout RBCs. For RNA-seq, a de novo assembly was conducted, in order to create a transcriptome database for RBCs. For proteome profiling, we developed a proteomic method that combined: (a) fractionation into cytosolic and membrane fractions, (b) hemoglobin removal of the cytosolic fraction, (c) protein digestion, and (d) a novel step with pH reversed-phase peptide fractionation and final Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC ESI-MS/MS) analysis of each fraction. Combined transcriptome- and proteome- sequencing data identified, in silico, novel and striking immune functional networks for rainbow trout nucleated RBCs, which are mainly linked to innate and adaptive immunity. Functional pathways related to regulation of hematopoietic cell differentiation, antigen presentation via major histocompatibility complex class II (MHCII), leukocyte differentiation and regulation of leukocyte activation were identified. These preliminary findings further implicate nucleated RBCs in immune function, such as antigen presentation and leukocyte activation.

Skeletal muscle proteomic signature and metabolic impairment in pulmonary hypertension.

Science.gov (United States)

Malenfant, Simon; Potus, François; Fournier, Frédéric; Breuils-Bonnet, Sandra; Pflieger, Aude; Bourassa, Sylvie; Tremblay, Ève; Nehmé, Benjamin; Droit, Arnaud; Bonnet, Sébastien; Provencher, Steeve

2015-05-01

Exercise limitation comes from a close interaction between cardiovascular and skeletal muscle impairments. To better understand the implication of possible peripheral oxidative metabolism dysfunction, we studied the proteomic signature of skeletal muscle in pulmonary arterial hypertension (PAH). Eight idiopathic PAH patients and eight matched healthy sedentary subjects were evaluated for exercise capacity, skeletal muscle proteomic profile, metabolism, and mitochondrial function. Skeletal muscle proteins were extracted, and fractioned peptides were tagged using an iTRAQ protocol. Proteomic analyses have documented a total of 9 downregulated proteins in PAH skeletal muscles and 10 upregulated proteins compared to healthy subjects. Most of the downregulated proteins were related to mitochondrial structure and function. Focusing on skeletal muscle metabolism and mitochondrial health, PAH patients presented a decreased expression of oxidative enzymes (pyruvate dehydrogenase, p metabolism in PAH skeletal muscles. We provide evidences that impaired mitochondrial and metabolic functions found in the lungs and the right ventricle are also present in skeletal muscles of patients. • Proteomic and metabolic analysis show abnormal oxidative metabolism in PAH skeletal muscle. • EM of PAH patients reveals abnormal mitochondrial structure and distribution. • Abnormal mitochondrial health and function contribute to exercise impairments of PAH. • PAH may be considered a vascular affliction of heart and lungs with major impact on peripheral muscles.

Murine colon proteome and characterization of the protein pathways

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Magdeldin Sameh

2012-08-01

Full Text Available Abstract Background Most of the current proteomic researches focus on proteome alteration due to pathological disorders (i.e.: colorectal cancer rather than normal healthy state when mentioning colon. As a result, there are lacks of information regarding normal whole tissue- colon proteome. Results We report here a detailed murine (mouse whole tissue- colon protein reference dataset composed of 1237 confident protein (FDR I and Mw ranged from 3–12 and 4–600 KDa, respectively. Gravy index scoring predicted 19.5% membranous and 80.5% globularly located proteins. GO hierarchies and functional network analysis illustrated proteins function together with their relevance and implication of several candidates in malignancy such as Mitogen- activated protein kinase (Mapk8, 9 in colorectal cancer, Fibroblast growth factor receptor (Fgfr 2, Glutathione S-transferase (Gstp1 in prostate cancer, and Cell division control protein (Cdc42, Ras-related protein (Rac1,2 in pancreatic cancer. Protein abundances calculated with 3 different algorithms (NSAF, PAF and emPAI provide a relative quantification under normal condition as guidance. Conclusions This highly confidence colon proteome catalogue will not only serve as a useful reference for further experiments characterizing differentially expressed proteins induced from diseased conditions, but also will aid in better understanding the ontology and functional absorptive mechanism of the colon as well.

Proteomics research in India: an update.

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Reddy, Panga Jaipal; Atak, Apurva; Ghantasala, Saicharan; Kumar, Saurabh; Gupta, Shabarni; Prasad, T S Keshava; Zingde, Surekha M; Srivastava, Sanjeeva

2015-09-08

After a successful completion of the Human Genome Project, deciphering the mystery surrounding the human proteome posed a major challenge. Despite not being largely involved in the Human Genome Project, the Indian scientific community contributed towards proteomic research along with the global community. Currently, more than 76 research/academic institutes and nearly 145 research labs are involved in core proteomic research across India. The Indian researchers have been major contributors in drafting the "human proteome map" along with international efforts. In addition to this, virtual proteomics labs, proteomics courses and remote triggered proteomics labs have helped to overcome the limitations of proteomics education posed due to expensive lab infrastructure. The establishment of Proteomics Society, India (PSI) has created a platform for the Indian proteomic researchers to share ideas, research collaborations and conduct annual conferences and workshops. Indian proteomic research is really moving forward with the global proteomics community in a quest to solve the mysteries of proteomics. A draft map of the human proteome enhances the enthusiasm among intellectuals to promote proteomic research in India to the world.This article is part of a Special Issue entitled: Proteomics in India. Copyright © 2015 Elsevier B.V. All rights reserved.

A draft map of the human ovarian proteome for tissue engineering and clinical applications.

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Ouni, Emna; Vertommen, Didier; Chiti, Maria Costanza; Dolmans, Marie-Madeleine; Amorim, Christiani Andrade

2018-02-23

Fertility preservation research in women today is increasingly taking advantage of bioengineering techniques to develop new biomimetic materials and solutions to safeguard ovarian cell function and microenvironment in vitro and in vivo. However, available data on the human ovary are limited and fundamental differences between animal models and humans are hampering researchers in their quest for more extensive knowledge of human ovarian physiology and key reproductive proteins that need to be preserved. We therefore turned to multi-dimensional label-free mass spectrometry to analyze human ovarian cortex, as it is a high-throughput and conclusive technique providing information on the proteomic composition of complex tissues like the ovary. In-depth proteomic profiling through two-dimensional liquid chromatography-mass spectrometry, western blot, histological and immunohistochemical analyses, and data mining helped us to confidently identify 1,508 proteins. Moreover, our method allowed us to chart the most complete representation so far of the ovarian matrisome, defined as the ensemble of extracellular matrix proteins and associated factors, including more than 80 proteins. In conclusion, this study will provide a better understanding of ovarian proteomics, with a detailed characterization of the ovarian follicle microenvironment, in order to enable bioengineers to create biomimetic scaffolds for transplantation and three-dimensional in vitro culture. By publishing our proteomic data, we also hope to contribute to accelerating biomedical research into ovarian health and disease in general. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Proteomic-Based Approaches for the Study of Cytokines in Lung Cancer

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Ángela Marrugal

2016-01-01

Full Text Available Proteomic techniques are currently used to understand the biology of different human diseases, including studies of the cell signaling pathways implicated in cancer progression, which is important in knowing the roles of different proteins in tumor development. Due to its poor prognosis, proteomic approaches are focused on the identification of new biomarkers for the early diagnosis, prognosis, and targeted treatment of lung cancer. Cytokines are proteins involved in inflammatory processes and have been proposed as lung cancer biomarkers and therapeutic targets because it has been reported that some cytokines play important roles in tumor development, invasion, and metastasis. In this review, we aim to summarize the different proteomic techniques used to discover new lung cancer biomarkers and therapeutic targets. Several cytokines have been identified as important players in lung cancer using these techniques. We underline the most important cytokines that are useful as biomarkers and therapeutic targets. We also summarize some of the therapeutic strategies targeted for these cytokines in lung cancer.

The core proteome and pan proteome of Salmonella Paratyphi A epidemic strains.

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Li Zhang

Full Text Available Comparative proteomics of the multiple strains within the same species can reveal the genetic variation and relationships among strains without the need to assess the genomic data. Similar to comparative genomics, core proteome and pan proteome can also be obtained within multiple strains under the same culture conditions. In this study we present the core proteome and pan proteome of four epidemic Salmonella Paratyphi A strains cultured under laboratory culture conditions. The proteomic information was obtained using a Two-dimensional gel electrophoresis (2-DE technique. The expression profiles of these strains were conservative, similar to the monomorphic genome of S. Paratyphi A. Few strain-specific proteins were found in these strains. Interestingly, non-core proteins were found in similar categories as core proteins. However, significant fluctuations in the abundance of some core proteins were also observed, suggesting that there is elaborate regulation of core proteins in the different strains even when they are cultured in the same environment. Therefore, core proteome and pan proteome analysis of the multiple strains can demonstrate the core pathways of metabolism of the species under specific culture conditions, and further the specific responses and adaptations of the strains to the growth environment.

Proteomic profiling of exosomes: Current perspectives

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Simpson, Richard J; Jensen, Søren S; Lim, Justin W E

2008-01-01

for the spread of morphogens in epithelia. The advent of current MS-based proteomic technologies has contributed significantly to our understanding of the molecular composition of exosomes. In addition to a common set of membrane and cytosolic proteins, it is becoming increasingly apparent that exosomes harbor...... distinct subsets of proteins that may be linked to cell-type associated functions. The secretion of exosomes by tumor cells and their implication in the transport and propagation of infectious cargo such as prions and retroviruses such as HIV suggest their participation in pathological situations...

Towards integrating extracellular matrix and immunological pathways.

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Boyd, David F; Thomas, Paul G

2017-10-01

The extracellular matrix (ECM) is a complex and dynamic structure made up of an estimated 300 different proteins. The ECM is also a rich source of cytokines and growth factors in addition to numerous bioactive ECM degradation products that influence cell migration, proliferation, and differentiation. The ECM is constantly being remodeled during homeostasis and in a wide range of pathological contexts. Changes in the ECM modulate immune responses, which in turn regulate repair and regeneration of tissues. Here, we review the many components of the ECM, enzymes involved in ECM remodeling, and the signals that feed into immunological pathways in the context of a dynamic ECM. We highlight studies that have taken an integrative approach to studying immune responses in the context of the ECM and studies that use novel proteomic strategies. Finally, we discuss research challenges relevant to the integration of immune and ECM networks and propose experimental and translational approaches to resolve these issues. Copyright © 2017 Elsevier Ltd. All rights reserved.

[Proteomics and transfusion medicine].

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Lion, N; Prudent, M; Crettaz, D; Tissot, J-D

2011-04-01

The term "proteomics" covers tools and techniques that are used to analyze and characterize complex mixtures of proteins from various biological samples. In this short review, a typical proteomic approach, related to the study of particular and illustrative situation related to transfusion medicine is reported. This "case report" will allow the reader to be familiar with a practical proteomic approach of a real situation, and will permit to describe the tools that are usually used in proteomic labs, and, in a second part, to present various proteomic applications in transfusion medicine. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Mistimed food intake and sleep alters 24-hour time-of-day patterns of the human plasma proteome.

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Depner, Christopher M; Melanson, Edward L; McHill, Andrew W; Wright, Kenneth P

2018-06-05

Proteomics holds great promise for understanding human physiology, developing health biomarkers, and precision medicine. However, how much the plasma proteome varies with time of day and is regulated by the master circadian suprachiasmatic nucleus brain clock, assessed here by the melatonin rhythm, is largely unknown. Here, we assessed 24-h time-of-day patterns of human plasma proteins in six healthy men during daytime food intake and nighttime sleep in phase with the endogenous circadian clock (i.e., circadian alignment) versus daytime sleep and nighttime food intake out of phase with the endogenous circadian clock (i.e., circadian misalignment induced by simulated nightshift work). We identified 24-h time-of-day patterns in 573 of 1,129 proteins analyzed, with 30 proteins showing strong regulation by the circadian cycle. Relative to circadian alignment, the average abundance and/or 24-h time-of-day patterns of 127 proteins were altered during circadian misalignment. Altered proteins were associated with biological pathways involved in immune function, metabolism, and cancer. Of the 30 circadian-regulated proteins, the majority peaked between 1400 hours and 2100 hours, and these 30 proteins were associated with basic pathways involved in extracellular matrix organization, tyrosine kinase signaling, and signaling by receptor tyrosine-protein kinase erbB-2. Furthermore, circadian misalignment altered multiple proteins known to regulate glucose homeostasis and/or energy metabolism, with implications for altered metabolic physiology. Our findings demonstrate the circadian clock, the behavioral wake-sleep/food intake-fasting cycle, and interactions between these processes regulate 24-h time-of-day patterns of human plasma proteins and help identify mechanisms of circadian misalignment that may contribute to metabolic dysregulation.

The Role of Extracellular Histones in Influenza Virus Pathogenesis.

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Ashar, Harshini K; Mueller, Nathan C; Rudd, Jennifer M; Snider, Timothy A; Achanta, Mallika; Prasanthi, Maram; Pulavendran, Sivasami; Thomas, Paul G; Ramachandran, Akhilesh; Malayer, Jerry R; Ritchey, Jerry W; Rajasekhar, Rachakatla; Chow, Vincent T K; Esmon, Charles T; Teluguakula, Narasaraju

2018-01-01

Although exaggerated host immune responses have been implicated in influenza-induced lung pathogenesis, the etiologic factors that contribute to these events are not completely understood. We previously demonstrated that neutrophil extracellular traps exacerbate pulmonary injury during influenza pneumonia. Histones are the major protein components of neutrophil extracellular traps and are known to have cytotoxic effects. Here, we examined the role of extracellular histones in lung pathogenesis during influenza. Mice infected with influenza virus displayed high accumulation of extracellular histones, with widespread pulmonary microvascular thrombosis. Occluded pulmonary blood vessels with vascular thrombi often exhibited endothelial necrosis surrounded by hemorrhagic effusions and pulmonary edema. Histones released during influenza induced cytotoxicity and showed strong binding to platelets within thrombi in infected mouse lungs. Nasal wash samples from influenza-infected patients also showed increased accumulation of extracellular histones, suggesting a possible clinical relevance of elevated histones in pulmonary injury. Although histones inhibited influenza growth in vitro, in vivo treatment with histones did not yield antiviral effects and instead exacerbated lung pathology. Blocking with antihistone antibodies caused a marked decrease in lung pathology in lethal influenza-challenged mice and improved protection when administered in combination with the antiviral agent oseltamivir. These findings support the pathogenic effects of extracellular histones in that pulmonary injury during influenza was exacerbated. Targeting histones provides a novel therapeutic approach to influenza pneumonia. Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Exosome proteomics reveals transcriptional regulator proteins with potential to mediate downstream pathways.

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Ung, Timothy H; Madsen, Helen J; Hellwinkel, Justin E; Lencioni, Alex M; Graner, Michael W

2014-11-01

Exosomes are virus-sized, membrane-enclosed vesicles with origins in the cellular endosomal system, but are released extracellularly. As a population, these tiny vesicles carry relatively enormous amounts of information in their protein, lipid and nucleic acid content, and the vesicles can have profound impacts on recipient cells. This review employs publically-available data combined with gene ontology applications to propose a novel concept, that exosomes transport transcriptional and translational machinery that may have direct impacts on gene expression in recipient cells. Here, we examine the previously published proteomic contents of medulloblastoma-derived exosomes, focusing on transcriptional regulators; we found that there are numerous proteins that may have potential roles in transcriptional and translational regulation with putative influence on downstream, cancer-related pathways. We expanded this search to all of the proteins in the Vesiclepedia database; using gene ontology approaches, we see that these regulatory factors are implicated in many of the processes involved in cancer initiation and progression. This information suggests that some of the effects of exosomes on recipient cells may be due to the delivery of protein factors that can directly and fundamentally change the transcriptional landscape of the cells. Within a tumor environment, this has potential to tilt the advantage towards the cancer. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Principles of proteome allocation are revealed using proteomic data and genome-scale models

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Yang, Laurence; Yurkovich, James T.; Lloyd, Colton J.

2016-01-01

to metabolism and fitness. Using proteomics data, we formulated allocation constraints for key proteome sectors in the ME model. The resulting calibrated model effectively computed the "generalist" (wild-type) E. coli proteome and phenotype across diverse growth environments. Across 15 growth conditions......Integrating omics data to refine or make context-specific models is an active field of constraint-based modeling. Proteomics now cover over 95% of the Escherichia coli proteome by mass. Genome-scale models of Metabolism and macromolecular Expression (ME) compute proteome allocation linked...... of these sectors for the general stress response sigma factor sigma(S). Finally, the sector constraints represent a general formalism for integrating omics data from any experimental condition into constraint-based ME models. The constraints can be fine-grained (individual proteins) or coarse-grained (functionally...

Attached and planktonic Listeria monocytogenes global proteomic responses and associated influence of strain genetics and temperature.

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Mata, Marcia M; da Silva, Wladimir P; Wilson, Richard; Lowe, Edwin; Bowman, John P

2015-02-06

Contamination of industrial and domestic food usage environments by the attachement of bacterial food-borne pathogen Listeria monocytogenes has public health and economic implications. Comprehensive proteomics experiments using label-free liquid chromatography/tandem mass spectrometry were used to compare the proteomes of two different L. monocytogenes strains (Siliken_1/2c and F2365_4b), which show very different capacities to attach to surfaces. Growth temperature and strain type were highly influential on the proteomes in both attached and planktonic cells. On the basis of the proteomic data, it is highly unlikely that specific surface proteins play a direct role in adherence to inanimate surfaces. Instead, strain-dependent responses related to cell envelope polymer biosynthesis and stress response regulation likely contribute to a different ability to attach and also to survive external stressors. Collectively, the divergent proteome-level responses observed define strain- and growth-temperature-dependent differences relevant to attachment efficacy, highlight relevant proteins involved in stress protection in attached cells, and suggest that strain differences and growth conditions are important in relation to environmental persistence.

In Silico Functional Networks Identified in Fish Nucleated Red Blood Cells by Means of Transcriptomic and Proteomic Profiling

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Sara Puente-Marin

2018-04-01

Full Text Available Nucleated red blood cells (RBCs of fish have, in the last decade, been implicated in several immune-related functions, such as antiviral response, phagocytosis or cytokine-mediated signaling. RNA-sequencing (RNA-seq and label-free shotgun proteomic analyses were carried out for in silico functional pathway profiling of rainbow trout RBCs. For RNA-seq, a de novo assembly was conducted, in order to create a transcriptome database for RBCs. For proteome profiling, we developed a proteomic method that combined: (a fractionation into cytosolic and membrane fractions, (b hemoglobin removal of the cytosolic fraction, (c protein digestion, and (d a novel step with pH reversed-phase peptide fractionation and final Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC ESI-MS/MS analysis of each fraction. Combined transcriptome- and proteome- sequencing data identified, in silico, novel and striking immune functional networks for rainbow trout nucleated RBCs, which are mainly linked to innate and adaptive immunity. Functional pathways related to regulation of hematopoietic cell differentiation, antigen presentation via major histocompatibility complex class II (MHCII, leukocyte differentiation and regulation of leukocyte activation were identified. These preliminary findings further implicate nucleated RBCs in immune function, such as antigen presentation and leukocyte activation.

Stressor-induced proteome alterations in zebrafish: A meta-analysis of response patterns

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Groh, Ksenia J., E-mail: ksenia.groh@eawag.ch [Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Dübendorf (Switzerland); ETH Zürich, Swiss Federal Institute of Technology, Department of Chemistry and Applied Biosciences, 8093 Zürich (Switzerland); Suter, Marc J.-F. [Eawag, Swiss Federal Institute of Aquatic Science and Technology, 8600 Dübendorf (Switzerland); ETH Zürich, Swiss Federal Institute of Technology, Department of Environmental Systems Science, 8092 Zürich (Switzerland)

2015-02-15

. We suggest that the results of any differential proteomics experiment performed with zebrafish should be interpreted keeping in mind the list of the most frequent responders that we have identified. Similar reservations should apply to any other species where proteome responses are analyzed by global proteomics methods. Careful consideration of the reliability and significance of observed changes is necessary in order not to over-interpret the experimental results and to prevent the proliferation of false positive linkages between the chemical and the cellular functions it perturbs. We further discuss the implications of the identified “top lists” of frequently responding proteins and protein families, and suggest further directions for proteomics research in ecotoxicology. Apart from improving the proteome coverage, further research should focus on defining the significance of the observed stress response patterns for organism phenotypes and on searching for common upstream regulators that can be targeted by specific assays.

Stressor-induced proteome alterations in zebrafish: A meta-analysis of response patterns

International Nuclear Information System (INIS)

Groh, Ksenia J.; Suter, Marc J.-F.

2015-01-01

. We suggest that the results of any differential proteomics experiment performed with zebrafish should be interpreted keeping in mind the list of the most frequent responders that we have identified. Similar reservations should apply to any other species where proteome responses are analyzed by global proteomics methods. Careful consideration of the reliability and significance of observed changes is necessary in order not to over-interpret the experimental results and to prevent the proliferation of false positive linkages between the chemical and the cellular functions it perturbs. We further discuss the implications of the identified “top lists” of frequently responding proteins and protein families, and suggest further directions for proteomics research in ecotoxicology. Apart from improving the proteome coverage, further research should focus on defining the significance of the observed stress response patterns for organism phenotypes and on searching for common upstream regulators that can be targeted by specific assays

Integrative analysis of the mitochondrial proteome in yeast.

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Holger Prokisch

2004-06-01

Full Text Available In this study yeast mitochondria were used as a model system to apply, evaluate, and integrate different genomic approaches to define the proteins of an organelle. Liquid chromatography mass spectrometry applied to purified mitochondria identified 546 proteins. By expression analysis and comparison to other proteome studies, we demonstrate that the proteomic approach identifies primarily highly abundant proteins. By expanding our evaluation to other types of genomic approaches, including systematic deletion phenotype screening, expression profiling, subcellular localization studies, protein interaction analyses, and computational predictions, we show that an integration of approaches moves beyond the limitations of any single approach. We report the success of each approach by benchmarking it against a reference set of known mitochondrial proteins, and predict approximately 700 proteins associated with the mitochondrial organelle from the integration of 22 datasets. We show that a combination of complementary approaches like deletion phenotype screening and mass spectrometry can identify over 75% of the known mitochondrial proteome. These findings have implications for choosing optimal genome-wide approaches for the study of other cellular systems, including organelles and pathways in various species. Furthermore, our systematic identification of genes involved in mitochondrial function and biogenesis in yeast expands the candidate genes available for mapping Mendelian and complex mitochondrial disorders in humans.

Extracellular peptidase hunting for improvement of protein production in plant cells and roots

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Jérôme eLallemand

2015-02-01

Full Text Available Plant-based recombinant protein production systems have gained an extensive interest over the past few years, because of their reduced cost and relative safety. Although the first products are now reaching the market, progress are still needed to improve plant hosts and strategies for biopharming. Targeting recombinant proteins toward the extracellular space offers several advantages in terms of protein folding and purification, but degradation events are observed, due to endogenous peptidases. This paper focuses on the analysis of extracellular proteolytic activities in two production systems: cell cultures and root-secretion (rhizosecretion, in Arabidopsis thaliana and Nicotiana tabacum. Proteolytic activities of extracellular proteomes (secretomes were evaluated in vitro against two substrate proteins: bovine serum albumin (BSA and human serum immunoglobulins G (hIgGs. Both targets were found to be degraded by the secretomes, BSA being more prone to proteolysis than hIgGs. The analysis of the proteolysis pH-dependence showed that target degradation was mainly dependent upon the production system: rhizosecretomes contained more peptidase activity than extracellular medium of cell suspensions, whereas variations due to plant species were smaller. Using class-specific peptidase inhibitors, serine and metallopeptidases were found to be responsible for degradation of both substrates. An in-depth in silico analysis of genomic and transcriptomic data from Arabidopsis was then performed and led to the identification of a limited number of serine and metallo-peptidases that are consistently expressed in both production systems. These peptidases should be prime candidates for further improvement of plant hosts by targeted silencing.

Specific extracellular matrix remodeling signature of colon hepatic metastases.

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Maguy Del Rio

Full Text Available To identify genes implicated in metastatic colonization of the liver in colorectal cancer, we collected pairs of primary tumors and hepatic metastases before chemotherapy in 13 patients. We compared mRNA expression in the pairs of patients to identify genes deregulated during metastatic evolution. We then validated the identified genes using data obtained by different groups. The 33-gene signature was able to classify 87% of hepatic metastases, 98% of primary tumors, 97% of normal colon mucosa, and 95% of normal liver tissues in six datasets obtained using five different microarray platforms. The identified genes are specific to colon cancer and hepatic metastases since other metastatic locations and hepatic metastases originating from breast cancer were not classified by the signature. Gene Ontology term analysis showed that 50% of the genes are implicated in extracellular matrix remodeling, and more precisely in cell adhesion, extracellular matrix organization and angiogenesis. Because of the high efficiency of the signature to classify colon hepatic metastases, the identified genes represent promising targets to develop new therapies that will specifically affect hepatic metastasis microenvironment.

In silico proteome analysis to facilitate proteomics experiments using mass spectrometry

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Lindo Micheal

2003-08-01

Full Text Available Abstract Proteomics experiments typically involve protein or peptide separation steps coupled to the identification of many hundreds to thousands of peptides by mass spectrometry. Development of methodology and instrumentation in this field is proceeding rapidly, and effective software is needed to link the different stages of proteomic analysis. We have developed an application, proteogest, written in Perl that generates descriptive and statistical analyses of the biophysical properties of multiple (e.g. thousands protein sequences submitted by the user, for instance protein sequences inferred from the complete genome sequence of a model organism. The application also carries out in silico proteolytic digestion of the submitted proteomes, or subsets thereof, and the distribution of biophysical properties of the resulting peptides is presented. proteogest is customizable, the user being able to select many options, for instance the cleavage pattern of the digestion treatment or the presence of modifications to specific amino acid residues. We show how proteogest can be used to compare the proteomes and digested proteome products of model organisms, to examine the added complexity generated by modification of residues, and to facilitate the design of proteomics experiments for optimal representation of component proteins.

The HUPO proteomics standards initiative--overcoming the fragmentation of proteomics data.

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Hermjakob, Henning

2006-09-01

Proteomics is a key field of modern biomolecular research, with many small and large scale efforts producing a wealth of proteomics data. However, the vast majority of this data is never exploited to its full potential. Even in publicly funded projects, often the raw data generated in a specific context is analysed, conclusions are drawn and published, but little attention is paid to systematic documentation, archiving, and public access to the data supporting the scientific results. It is often difficult to validate the results stated in a particular publication, and even simple global questions like "In which cellular contexts has my protein of interest been observed?" can currently not be answered with realistic effort, due to a lack of standardised reporting and collection of proteomics data. The Proteomics Standards Initiative (PSI), a work group of the Human Proteome Organisation (HUPO), defines community standards for data representation in proteomics to facilitate systematic data capture, comparison, exchange and verification. In this article we provide an overview of PSI organisational structure, activities, and current results, as well as ways to get involved in the broad-based, open PSI process.

Making proteomics data accessible and reusable: current state of proteomics databases and repositories.

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Perez-Riverol, Yasset; Alpi, Emanuele; Wang, Rui; Hermjakob, Henning; Vizcaíno, Juan Antonio

2015-03-01

Compared to other data-intensive disciplines such as genomics, public deposition and storage of MS-based proteomics, data are still less developed due to, among other reasons, the inherent complexity of the data and the variety of data types and experimental workflows. In order to address this need, several public repositories for MS proteomics experiments have been developed, each with different purposes in mind. The most established resources are the Global Proteome Machine Database (GPMDB), PeptideAtlas, and the PRIDE database. Additionally, there are other useful (in many cases recently developed) resources such as ProteomicsDB, Mass Spectrometry Interactive Virtual Environment (MassIVE), Chorus, MaxQB, PeptideAtlas SRM Experiment Library (PASSEL), Model Organism Protein Expression Database (MOPED), and the Human Proteinpedia. In addition, the ProteomeXchange consortium has been recently developed to enable better integration of public repositories and the coordinated sharing of proteomics information, maximizing its benefit to the scientific community. Here, we will review each of the major proteomics resources independently and some tools that enable the integration, mining and reuse of the data. We will also discuss some of the major challenges and current pitfalls in the integration and sharing of the data. © 2014 The Authors. PROTEOMICS published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The proteomic complexity and rise of the primordial ancestor of diversified life

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Kim Kyung

2011-05-01

biosynthesis of membrane sn1,2 glycerol ester and ether lipids, and had crucial elements of translation, including a primordial ribosome with protein synthesis capabilities. It lacked however fundamental functions, including transcription, processes for extracellular communication, and enzymes for deoxyribonucleotide synthesis. Proteomic history reveals the urancestor is closer to a simple progenote organism but harbors a rather complex set of modern molecular functions.

Recent Advances in the Composition and Heterogeneity of the Arabidopsis Mitochondrial Proteome

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Chun Pong eLee

2013-01-01

Full Text Available Mitochondria are important organelles for providing the ATP and carbon skeletons required to sustain cell growth. While these organelles also participate in other key metabolic functions across species, they have a specialized role in plants of optimizing photosynthesis through participating in photorespiration. It is therefore critical to map the protein composition of mitochondria in plants to gain a better understanding of their regulation and define the uniqueness of their metabolic networks. To date, less than 30% of the predicted number of mitochondrial proteins has been verified experimentally by proteomics and/or GFP localization studies. In this mini-review, we will provide an overview of the advances in mitochondrial proteomics in the model plant Arabidopsis thaliana over the past five years. The ultimate goal of mapping the mitochondrial proteome in Arabidopsis is to discover novel mitochondrial components that are critical during development in plants as well as genes involved in developmental abnormalities, such as those implicated in mitochondrial-linked cytoplasmic male sterility.

Skeletal muscle expresses the extracellular cyclic AMP–adenosine pathway

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Chiavegatti, T; Costa, V L; Araújo, M S; Godinho, R O

2007-01-01

Background and purpose: cAMP is a key intracellular signalling molecule that regulates multiple processes of the vertebrate skeletal muscle. We have shown that cAMP can be actively pumped out from the skeletal muscle cell. Since in other tissues, cAMP efflux had been associated with extracellular generation of adenosine, in the present study we have assessed the fate of interstitial cAMP and the existence of an extracellular cAMP-adenosine signalling pathway in skeletal muscle. Experimental approach: cAMP efflux and/or its extracellular degradation were analysed by incubating rat cultured skeletal muscle with exogenous cAMP, forskolin or isoprenaline. cAMP and its metabolites were quantified by radioassay or HPLC, respectively. Key results: Incubation of cells with exogenous cAMP was followed by interstitial accumulation of 5′-AMP and adenosine, a phenomenon inhibited by selective inhibitors of ecto-phosphodiesterase (DPSPX) and ecto-nucleotidase (AMPCP). Activation of adenylyl cyclase (AC) in cultured cells with forskolin or isoprenaline increased cAMP efflux and extracellular generation of 5′-AMP and adenosine. Extracellular cAMP-adenosine pathway was also observed after direct and receptor-dependent stimulation of AC in rat extensor muscle ex vivo. These events were attenuated by probenecid, an inhibitor of ATP binding cassette family transporters. Conclusions and implications: Our results show the existence of an extracellular biochemical cascade that converts cAMP into adenosine. The functional relevance of this extracellular signalling system may involve a feedback modulation of cellular response initiated by several G protein-coupled receptor ligands, amplifying cAMP influence to a paracrine mode, through its metabolite, adenosine. PMID:18157164

Personalized medicine beyond genomics: alternative futures in big data-proteomics, environtome and the social proteome.

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Özdemir, Vural; Dove, Edward S; Gürsoy, Ulvi K; Şardaş, Semra; Yıldırım, Arif; Yılmaz, Şenay Görücü; Ömer Barlas, I; Güngör, Kıvanç; Mete, Alper; Srivastava, Sanjeeva

2017-01-01

No field in science and medicine today remains untouched by Big Data, and psychiatry is no exception. Proteomics is a Big Data technology and a next generation biomarker, supporting novel system diagnostics and therapeutics in psychiatry. Proteomics technology is, in fact, much older than genomics and dates to the 1970s, well before the launch of the international Human Genome Project. While the genome has long been framed as the master or "elite" executive molecule in cell biology, the proteome by contrast is humble. Yet the proteome is critical for life-it ensures the daily functioning of cells and whole organisms. In short, proteins are the blue-collar workers of biology, the down-to-earth molecules that we cannot live without. Since 2010, proteomics has found renewed meaning and international attention with the launch of the Human Proteome Project and the growing interest in Big Data technologies such as proteomics. This article presents an interdisciplinary technology foresight analysis and conceptualizes the terms "environtome" and "social proteome". We define "environtome" as the entire complement of elements external to the human host, from microbiome, ambient temperature and weather conditions to government innovation policies, stock market dynamics, human values, political power and social norms that collectively shape the human host spatially and temporally. The "social proteome" is the subset of the environtome that influences the transition of proteomics technology to innovative applications in society. The social proteome encompasses, for example, new reimbursement schemes and business innovation models for proteomics diagnostics that depart from the "once-a-life-time" genotypic tests and the anticipated hype attendant to context and time sensitive proteomics tests. Building on the "nesting principle" for governance of complex systems as discussed by Elinor Ostrom, we propose here a 3-tiered organizational architecture for Big Data science such as

Differential effect of TGFβ on the proteome of cancer associated fibroblasts and cancer epithelial cells in a co-culture approach - a short report.

Science.gov (United States)

Koczorowska, Maria Magdalena; Friedemann, Charlotte; Geiger, Klaus; Follo, Marie; Biniossek, Martin Lothar; Schilling, Oliver

2017-12-01

Solid tumors contain various components that together form the tumor microenvironment. Cancer associated fibroblasts (CAFs) are capable of secreting and responding to signaling molecules and growth factors. Due to their role in tumor development, CAFs are considered as potential therapeutic targets. A prominent tumor-associated signaling molecule is transforming growth factor β (TGFβ), an inducer of epithelial-to-mesenchymal transition (EMT). The differential action of TGFβ on CAFs and ETCs (epithelial tumor cells) has recently gained interest. Here, we aimed to investigate the effects of TGFβ on CAFs and ETCs at the proteomic level. We established a 2D co-culture system of differentially fluorescently labeled CAFs and ETCs and stimulated this co-culture system with TGFβ. The respective cell types were separated using FACS and subjected to quantitative analyses of individual proteomes using mass spectrometry. We found that TGFβ treatment had a strong impact on the proteome composition of CAFs, whereas ETCs responded only marginally to TGFβ. Quantitative proteomic analyses of the different cell types revealed up-regulation of extracellular matrix (ECM) proteins in TGFβ treated CAFs. In addition, we found that the TGFβ treated CAFs exhibited increased N-cadherin levels. From our data we conclude that CAFs respond to TGFβ treatment by changing their proteome composition, while ETCs appear to be rather resilient.

Detection of Nuclear Protein Profile Changes by Human Metapneumovirus M2-2 Protein Using Quantitative Differential Proteomics

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Yuping Ren

2017-12-01

Full Text Available Human metapneumovirus (hMPV is a leading cause of lower respiratory infection in pediatric populations globally. This study examined proteomic profile changes in A549 cells infected with hMPV and two attenuated mutants with deleted PDZ domain-binding motif(s in the M2-2 protein. These motifs are involved in the interruption of antiviral signaling, namely the interaction between the TNF receptor associated factor (TRAF and mitochondrial antiviral-signaling (MAVS proteins. The aim of this study was to provide insight into the overall and novel impact of M2-2 motifs on cellular responses via an unbiased comparison. Tandem mass tagging, stable isotope labeling, and high-resolution mass spectrometry were used for quantitative proteomic analysis. Using quantitative proteomics and Venn analysis, 1248 common proteins were detected in all infected samples of both technical sets. Hierarchical clustering of the differentiated proteome displayed distinct proteomic signatures that were controlled by the motif(s. Bioinformatics and experimental analysis confirmed the differentiated proteomes, revealed novel cellular biological events, and implicated key pathways controlled by hMPV M2-2 PDZ domain-binding motif(s. This provides further insight for evaluating M2-2 mutants as potent vaccine candidates.

The extracellular proteome of Bacillus licheniformis grown in different media and under different nutrient starvation conditions

NARCIS (Netherlands)

Voigt, B; Schweder, T; Sibbald, MJJB; Albrecht, D; Ehrenreich, A; Bernhardt, J; Feesche, J; Maurer, KH; Gottschalk, G; van Dijl, JM; Hecker, M

The now finished genome sequence of Bacillus licheniformis DSM 13 allows the prediction of the genes involved in protein secretion into the extracellular environment as well as the prediction of the proteins which are translocated. From the sequence 296 proteins were predicted to contain an

Proteomics in medical microbiology.

Science.gov (United States)

Cash, P

2000-04-01

The techniques of proteomics (high resolution two-dimensional electrophoresis and protein characterisation) are widely used for microbiological research to analyse global protein synthesis as an indicator of gene expression. The rapid progress in microbial proteomics has been achieved through the wide availability of whole genome sequences for a number of bacterial groups. Beyond providing a basic understanding of microbial gene expression, proteomics has also played a role in medical areas of microbiology. Progress has been made in the use of the techniques for investigating the epidemiology and taxonomy of human microbial pathogens, the identification of novel pathogenic mechanisms and the analysis of drug resistance. In each of these areas, proteomics has provided new insights that complement genomic-based investigations. This review describes the current progress in these research fields and highlights some of the technical challenges existing for the application of proteomics in medical microbiology. The latter concern the analysis of genetically heterogeneous bacterial populations and the integration of the proteomic and genomic data for these bacteria. The characterisation of the proteomes of bacterial pathogens growing in their natural hosts remains a future challenge.

Proteomics identifies Bacillus cereus EntD as a pivotal protein for the production of numerous virulence factors

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Hélène eOmer

2015-10-01

Full Text Available Bacillus cereus is a gram-positive pathogen that causes a wide variety of diseases in humans. It secretes into the extracellular milieu proteins that may contribute directly or indirectly to its virulence. EntD is a novel exoprotein identified by proteogenomics of B. cereus ATCC 14579. We constructed a ΔentD mutant and analyzed the impact of entD disruption on the cellular proteome and exoproteome isolated from early, late and stationary-phase cultures. We identified 308 and 79 proteins regulated by EntD in the cellular proteome and the exoproteome, respectively. The contribution of these proteins to important virulence-associated functions, including central metabolism, cell structure, antioxidative ability, cell motility and toxin production, are presented. The proteomic data were correlated with the growth defect, cell morphology change, reduced motility and reduced cytotoxicity of the ΔentD mutant strain. We conclude that EntD is an important player in B. cereus virulence. The function of EntD and the putative EntD-dependent regulatory network are discussed. To our knowledge, this study is the first characterization of an Ent family protein in a species of the B. cereus group.

The iron-responsive microsomal proteome of Aspergillus fumigatus.

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Moloney, Nicola M; Owens, Rebecca A; Meleady, Paula; Henry, Michael; Dolan, Stephen K; Mulvihill, Eoin; Clynes, Martin; Doyle, Sean

2016-03-16

Aspergillus fumigatus is an opportunistic fungal pathogen. Siderophore biosynthesis and iron acquisition are essential for virulence. Yet, limited data exist with respect to the adaptive nature of the fungal microsomal proteome under iron-limiting growth conditions, as encountered during host infection. Here, we demonstrate that under siderophore biosynthetic conditions--significantly elevated fusarinine C (FSC) and triacetylfusarinine C (TAFC) production (pproteome remodelling occurs. Specifically, a four-fold enrichment of transmembrane-containing proteins was observed with respect to whole cell lysates following ultracentrifugation-based microsomal extraction. Comparative label-free proteomic analysis of microsomal extracts, isolated following iron-replete and -deplete growth, identified 710 unique proteins. Scatterplot analysis (MaxQuant) demonstrated high correlation amongst biological replicates from each growth condition (Pearson correlation >0.96 within groups; biological replicates (n=4)). Quantitative and qualitative comparison revealed 231 proteins with a significant change in abundance between the iron-replete and iron-deplete conditions (pAspergillus fumigatus must acquire iron to facilitate growth and pathogenicity. Iron-chelating non-ribosomal peptides, termed siderophores, mediate iron uptake via membrane-localised transporter proteins. Here we demonstrate for the first time that growth of A. fumigatus under iron-deplete conditions, concomitant with siderophore biosynthesis, leads to an extensive remodelling of the microsomal proteome which includes significantly altered levels of 231 constituent proteins (96 increased and 135 decreased in abundance), many of which have not previously been localised to the microsome. We also demonstrate the first synthesis of a fluorescent version of fusarinine C, an extracellular A. fumigatus siderophore, and its uptake and localization under iron-restricted conditions. This infers the use of an A. fumigatus

OralCard: a bioinformatic tool for the study of oral proteome.

Science.gov (United States)

Arrais, Joel P; Rosa, Nuno; Melo, José; Coelho, Edgar D; Amaral, Diana; Correia, Maria José; Barros, Marlene; Oliveira, José Luís

2013-07-01

The molecular complexity of the human oral cavity can only be clarified through identification of components that participate within it. However current proteomic techniques produce high volumes of information that are dispersed over several online databases. Collecting all of this data and using an integrative approach capable of identifying unknown associations is still an unsolved problem. This is the main motivation for this work. We present the online bioinformatic tool OralCard, which comprises results from 55 manually curated articles reflecting the oral molecular ecosystem (OralPhysiOme). It comprises experimental information available from the oral proteome both of human (OralOme) and microbial origin (MicroOralOme) structured in protein, disease and organism. This tool is a key resource for researchers to understand the molecular foundations implicated in biology and disease mechanisms of the oral cavity. The usefulness of this tool is illustrated with the analysis of the oral proteome associated with diabetes melitus type 2. OralCard is available at http://bioinformatics.ua.pt/oralcard. Copyright © 2013 Elsevier Ltd. All rights reserved.

Method for isolation and molecular characterization of extracellular microvesicles released from brain endothelial cells

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Haqqani Arsalan S

2013-01-01

Full Text Available Abstract Background In addition to possessing intracellular vesicles, eukaryotic cells also produce extracellular microvesicles, ranging from 50 to 1000 nm in diameter that are released or shed into the microenvironment under physiological and pathological conditions. These membranous extracellular organelles include both exosomes (originating from internal vesicles of endosomes and ectosomes (originating from direct budding/shedding of plasma membranes. Extracellular microvesicles contain cell-specific collections of proteins, glycoproteins, lipids, nucleic acids and other molecules. These vesicles play important roles in intercellular communication by acting as carrier for essential cell-specific information to target cells. Endothelial cells in the brain form the blood–brain barrier, a specialized interface between the blood and the brain that tightly controls traffic of nutrients and macromolecules between two compartments and interacts closely with other cells forming the neurovascular unit. Therefore, brain endothelial cell extracellular microvesicles could potentially play important roles in ‘externalizing’ brain-specific biomarkers into the blood stream during pathological conditions, in transcytosis of blood-borne molecules into the brain, and in cell-cell communication within the neurovascular unit. Methods To study cell-specific molecular make-up and functions of brain endothelial cell exosomes, methods for isolation of extracellular microvesicles using mass spectrometry-compatible protocols and the characterization of their signature profiles using mass spectrometry -based proteomics were developed. Results A total of 1179 proteins were identified in the isolated extracellular microvesicles from brain endothelial cells. The microvesicles were validated by identification of almost 60 known markers, including Alix, TSG101 and the tetraspanin proteins CD81 and CD9. The surface proteins on isolated microvesicles could potentially

Proteomic Characterization of Primary Mouse Hepatocytes in Collagen Monolayer and Sandwich Culture.

Science.gov (United States)

Orsini, Malina; Sperber, Saskia; Noor, Fozia; Hoffmann, Esther; Weber, Susanne N; Hall, Rabea A; Lammert, Frank; Heinzle, Elmar

2018-01-01

Dedifferentiation of primary hepatocytes in vitro makes their application in long-term studies difficult. Embedding hepatocytes in a sandwich of extracellular matrix is reported to delay the dedifferentiation process to some extent. In this study, we compared the intracellular proteome of primary mouse hepatocytes (PMH) in conventional monolayer cultures (ML) to collagen sandwich culture (SW) after 1 day and 5 days of cultivation. Quantitative proteome analysis of PMH showed no differences between collagen SW and ML cultures after 1 day. Glycolysis and gluconeogenesis were strongly affected by long-term cultivation in both ML and SW cultures. Interestingly, culture conditions had no effect on cellular lipid metabolism. After 5 days, PMH in collagen SW and ML cultures exhibit characteristic indications of oxidative stress. However, in the SW culture the defense system against oxidative stress is significantly up-regulated to deal with this, whereas in the ML culture a down-regulation of these important enzymes takes place. Regarding the multiple effects of ROS and oxidative stress in cells, we conclude that the down-regulation of these enzymes seem to play a role in the loss of hepatic function observed in the ML cultivation. In addition, enzymes of the urea cycle were clearly down-regulated in ML culture. Proteomics confirms lack in oxidative stress defense mechanisms as the major characteristic of hepatocytes in monolayer cultures compared to sandwich cultures. J. Cell. Biochem. 119: 447-454, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Data from proteomic characterization and comparison of mammalian milk fat globule proteomes by iTRAQ analysis

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Yongxin Yang

2015-06-01

Full Text Available Milk fat globules memebrane (MFGM-enriched proteomes from Holstein, Jersey, yak, buffalo, goat, camel, horse, and human were extracted and identified by an iTRAQ quantification proteomic approach. Proteomes data were analyzed by bioinformatic and multivariate statistical analysis and used to present the characteristic traits of the MFGM proteins among the studied mammals. The data of this study are also related to the research article “Proteomic characterization and comparison of mammalian milk fat globule proteomes by iTRAQ analysis” in the Journal of Proteomics [1].

An experimental and bioinformatic tissue proteomics-strategy applied to human hepatocellular carcinoma focussing on functional data interpretation

International Nuclear Information System (INIS)

Zwickl, H.

2010-01-01

The main challenge of tissue proteomics arises from the intrinsic complexity of samples which impedes reliable data interpretation. Tissues are composed of different cell types and a specific extracellular matrix. Although tumors are supposed to arise from a single cell type (e.g. HCC from hepatocytes or liver stem cells), their composition is additionally influenced by e.g. the infiltration of immune cells, by changes in the abundance of cells constituting also non-tumorous tissues and by epithelial-mesenchymal transition of cells due to an increase of developmental plasticity and loss of differentiation state usually accompanying tumor progression as well as the extracellular matrix as crucial constituent of the tumor microenvironment. The aim of this work was to determine functional differences of non-tumorous liver and HCC tissue via proteomics methods (shotgun, mass spectrometry, 2D-PAGE). For data interpretation, the focus was laid on basic cell biological and metabolic alterations resulting in the loss or gain of functions rather than those occuring at the single gene (protein) level. Proteins were functionally clustered based on several databases (e.g. KEGG, PubMed) as well as biochemical and toxicological literature. Moreover, functional proteome alterations were correlated to physiologically, histologically, and cytologically observable alterations. In order to enable the comparison of immunohistochemically (from www.proteinatlas.org) and tissue proteomics-derived protein expression, a method for the elaboration of histologic data was introduced. In addition, proteome data were compared to those of 'Encyclopedia of Hepatocellular Carcinoma genes Online' (EHCO), a database compiling eight gene set collections including PubMed, SAGE, microarray, and proteomics data. HCC tissues generally exhibit reduced secretion performance and concomitantly enhanced cytoplasmic protein synthesis (see paper 'A novel technique to specifically analyze the secretome of cells

Bacterial membrane proteomics.

Science.gov (United States)

Poetsch, Ansgar; Wolters, Dirk

2008-10-01

About one quarter to one third of all bacterial genes encode proteins of the inner or outer bacterial membrane. These proteins perform essential physiological functions, such as the import or export of metabolites, the homeostasis of metal ions, the extrusion of toxic substances or antibiotics, and the generation or conversion of energy. The last years have witnessed completion of a plethora of whole-genome sequences of bacteria important for biotechnology or medicine, which is the foundation for proteome and other functional genome analyses. In this review, we discuss the challenges in membrane proteome analysis, starting from sample preparation and leading to MS-data analysis and quantification. The current state of available proteomics technologies as well as their advantages and disadvantages will be described with a focus on shotgun proteomics. Then, we will briefly introduce the most abundant proteins and protein families present in bacterial membranes before bacterial membrane proteomics studies of the last years will be presented. It will be shown how these works enlarged our knowledge about the physiological adaptations that take place in bacteria during fine chemical production, bioremediation, protein overexpression, and during infections. Furthermore, several examples from literature demonstrate the suitability of membrane proteomics for the identification of antigens and different pathogenic strains, as well as the elucidation of membrane protein structure and function.

Finding a Leucine in a Haystack: Searching the Proteome for ambigous Leucine-Aspartic Acid motifs

KAUST Repository

Arold, Stefan T.

2016-01-01

LDMF predicted 13 new LD motifs in humans. Using biophysical assays, we experimentally confirmed in vitro interactions for four novel LD motif proteins. Thus, LDMF allows proteome-wide discovery of LD motifs, despite a highly ambiguous sequence pattern. Functional implications will be discussed.

Comprehensive proteome analysis of nasal lavage samples after controlled exposure to welding nanoparticles shows an induced acute phase and a nuclear receptor, LXR/RXR, activation that influence the status of the extracellular matrix.

Science.gov (United States)

Ali, Neserin; Ljunggren, Stefan; Karlsson, Helen M; Wierzbicka, Aneta; Pagels, Joakim; Isaxon, Christina; Gudmundsson, Anders; Rissler, Jenny; Nielsen, Jörn; Lindh, Christian H; Kåredal, Monica

2018-01-01

results suggested that the exposure causes an immediate effect on the proteome level involving acute phase proteins and mediators regulating lipid signaling. Proteases involved in maintaining the balance between the formation and degradation of extracellular matrix proteins are important key proteins in the induced effects.

[Diet and exercise influence on the proteomic profile of an athlete population].

Science.gov (United States)

Toro, Rocio; Mangas, Alipio; Quezada, Maribel; Rodriguez-Rosety, Manuel; Fournielles, Gabriel; Rodriguez-Rosety, Ignacio; Rodriguez Rosety, Miguel Angel; Alonso, Jose Angel; Garcia-Cozar, Francisco Jose; Duran, Maria Del Carmen

2014-11-01

Nutrition has emerged as a fundamental tool included in the training program of athletes. Body composition seeks different objectives depending on type of sport, position, or time of the season. Furthermore, analysis proteomics allows us to know the structure and function of proteins. To study, using proteomics, the influence of two different diets on the anthropometric profile in a rugby players group. It is a prospective and interventionist study. Thirty-two rugby players were included. Two groups were defined, one followed proteic diet (PD) and, the other group subscribed the Mediterranean diet (MD). All participants were evaluated anthropometrically at the beginning and after six months. A blood sample was taken to twenty -two players, half of each group, used for the proteomic analysis. MD highlight more benefit for these athletes. Two groups were defined based on their anthropometric behavior, G1 and G2. The proteomic analysis related significantly some TGF-family mediators with these groups. MD improves the muscular mass without increasing the total body weight, so this data could be determinant to define profiles for athletes. Some TGF-members could be implicated in the adipose tissue and muscular mass balance. Copyright AULA MEDICA EDICIONES 2014. Published by AULA MEDICA. All rights reserved.

Proteomics reveals the effects of sustained weight loss on the human plasma proteome

DEFF Research Database (Denmark)

Geyer, Philipp E; Wewer Albrechtsen, Nicolai J; Tyanova, Stefka

2016-01-01

Sustained weight loss is a preferred intervention in a wide range of metabolic conditions, but the effects on an individual's health state remain ill-defined. Here, we investigate the plasma proteomes of a cohort of 43 obese individuals that had undergone 8 weeks of 12% body weight loss followed...... by a year of weight maintenance. Using mass spectrometry-based plasma proteome profiling, we measured 1,294 plasma proteomes. Longitudinal monitoring of the cohort revealed individual-specific protein levels with wide-ranging effects of losing weight on the plasma proteome reflected in 93 significantly...

Translational plant proteomics: a perspective.

Science.gov (United States)

Agrawal, Ganesh Kumar; Pedreschi, Romina; Barkla, Bronwyn J; Bindschedler, Laurence Veronique; Cramer, Rainer; Sarkar, Abhijit; Renaut, Jenny; Job, Dominique; Rakwal, Randeep

2012-08-03

Translational proteomics is an emerging sub-discipline of the proteomics field in the biological sciences. Translational plant proteomics aims to integrate knowledge from basic sciences to translate it into field applications to solve issues related but not limited to the recreational and economic values of plants, food security and safety, and energy sustainability. In this review, we highlight the substantial progress reached in plant proteomics during the past decade which has paved the way for translational plant proteomics. Increasing proteomics knowledge in plants is not limited to model and non-model plants, proteogenomics, crop improvement, and food analysis, safety, and nutrition but to many more potential applications. Given the wealth of information generated and to some extent applied, there is the need for more efficient and broader channels to freely disseminate the information to the scientific community. This article is part of a Special Issue entitled: Translational Proteomics. Copyright © 2012 Elsevier B.V. All rights reserved.

Transformative Impact of Proteomics on Cardiovascular Health and Disease: A Scientific Statement From the American Heart Association.

Science.gov (United States)

Lindsey, Merry L; Mayr, Manuel; Gomes, Aldrin V; Delles, Christian; Arrell, D Kent; Murphy, Anne M; Lange, Richard A; Costello, Catherine E; Jin, Yu-Fang; Laskowitz, Daniel T; Sam, Flora; Terzic, Andre; Van Eyk, Jennifer; Srinivas, Pothur R

2015-09-01

The year 2014 marked the 20th anniversary of the coining of the term proteomics. The purpose of this scientific statement is to summarize advances over this period that have catalyzed our capacity to address the experimental, translational, and clinical implications of proteomics as applied to cardiovascular health and disease and to evaluate the current status of the field. Key successes that have energized the field are delineated; opportunities for proteomics to drive basic science research, facilitate clinical translation, and establish diagnostic and therapeutic healthcare algorithms are discussed; and challenges that remain to be solved before proteomic technologies can be readily translated from scientific discoveries to meaningful advances in cardiovascular care are addressed. Proteomics is the result of disruptive technologies, namely, mass spectrometry and database searching, which drove protein analysis from 1 protein at a time to protein mixture analyses that enable large-scale analysis of proteins and facilitate paradigm shifts in biological concepts that address important clinical questions. Over the past 20 years, the field of proteomics has matured, yet it is still developing rapidly. The scope of this statement will extend beyond the reaches of a typical review article and offer guidance on the use of next-generation proteomics for future scientific discovery in the basic research laboratory and clinical settings. © 2015 American Heart Association, Inc.

Proteomics in Argentina - limitations and future perspectives: A special emphasis on meat proteomics.

Science.gov (United States)

Fadda, Silvina; Almeida, André M

2015-11-01

Argentina is one of the most relevant countries in Latin America, playing a major role in regional economics, culture and science. Over the last 80 years, Argentinean history has been characterized by several upward and downward phases that had major consequences on the development of science in the country and most recently on proteomics. In this article, we characterize the evolution of Proteomics sciences in Argentina over the last decade and a half. We describe the proteomics publication output of the country in the framework of the regional and international contexts, demonstrating that Argentina is solidly anchored in a regional context, showing results similar to other emergent and Latin American countries, albeit still far from the European, American or Australian realities. We also provide a case-study on the importance of Proteomics to a specific sector in the area of food science: the use of bacteria of technological interest, highlighting major achievements obtained by Argentinean proteomics scientists. Finally, we provide a general picture of the endeavors being undertaken by Argentinean Proteomics scientists and their international collaborators to promote the Proteomics-based research with the new generation of scientists and PhD students in both Argentina and other countries in the Southern cone. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biochemical Markers of Brain Injury: An Integrated Proteomics-Based Approach

Science.gov (United States)

2006-02-01

some variability between protein assay measurements. Using densitometric analysis after 1D-PAGE, microfiltration alone showed an 11 ( 5% sample reduction...KOPETSCH, O., WOSZCZYK, A., et al. (2003). Serum S-100B protein as a molecular marker in severe trau- matic brain injury. Restor . Neurol. Neurosci. 21...proteomics. Implications in the search for preventive initiatives to slow the clinical progression of Alzheimer’s disease dementia. Restor . Neurol. Neurosci

Label-Free LC-MS/MS Proteomic Analysis of Cerebrospinal Fluid Identifies Protein/Pathway Alterations and Candidate Biomarkers for Amyotrophic Lateral Sclerosis.

Science.gov (United States)

Collins, Mahlon A; An, Jiyan; Hood, Brian L; Conrads, Thomas P; Bowser, Robert P

2015-11-06

Analysis of the cerebrospinal fluid (CSF) proteome has proven valuable to the study of neurodegenerative disorders. To identify new protein/pathway alterations and candidate biomarkers for amyotrophic lateral sclerosis (ALS), we performed comparative proteomic profiling of CSF from sporadic ALS (sALS), healthy control (HC), and other neurological disease (OND) subjects using label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 1712 CSF proteins were detected and relatively quantified by spectral counting. Levels of several proteins with diverse biological functions were significantly altered in sALS samples. Enrichment analysis was used to link these alterations to biological pathways, which were predominantly related to inflammation, neuronal activity, and extracellular matrix regulation. We then used our CSF proteomic profiles to create a support vector machines classifier capable of discriminating training set ALS from non-ALS (HC and OND) samples. Four classifier proteins, WD repeat-containing protein 63, amyloid-like protein 1, SPARC-like protein 1, and cell adhesion molecule 3, were identified by feature selection and externally validated. The resultant classifier distinguished ALS from non-ALS samples with 83% sensitivity and 100% specificity in an independent test set. Collectively, our results illustrate the utility of CSF proteomic profiling for identifying ALS protein/pathway alterations and candidate disease biomarkers.

Proteomic analysis of astrocytic secretion that regulates neurogenesis using quantitative amine-specific isobaric tagging

Energy Technology Data Exchange (ETDEWEB)

Yan, Hu; Zhou, Wenhao [Children' s Hospital of Fudan University, 399 Wanyuan Road, Shanghai 201102 (China); Wei, Liming; Zhong, Fan [Institutes of Biomedical Sciences, Fudan University, 138 Yixueyuan Roda, Shanghai 200032 (China); Yang, Yi, E-mail: yyang@shmu.edu.cn [Children' s Hospital of Fudan University, 399 Wanyuan Road, Shanghai 201102 (China)

2010-01-08

Astrocytes are essential components of neurogenic niches that affect neurogenesis through membrane association and/or the release of soluble factors. To identify factors released from astrocytes that could regulate neural stem cell differentiation and proliferation, we used mild oxygen-glucose deprivation (OGD) to inhibit the secretory capacity of astrocytes. Using the Transwell co-culture system, we found that OGD-treated astrocytes could not promote neural stem cell differentiation and proliferation. Next, isobaric tagging for the relative and absolute quantitation (iTRAQ) proteomics techniques was performed to identify the proteins in the supernatants of astrocytes (with or without OGD). Through a multi-step analysis and gene ontology classification, 130 extracellular proteins were identified, most of which were involved in neuronal development, the inflammatory response, extracellular matrix composition and supportive functions. Of these proteins, 44 had never been reported to be produced by astrocytes. Using ProteinPilot software analysis, we found that 60 extracellular proteins were significantly altered (27 upregulated and 33 downregulated) in the supernatant of OGD-treated astrocytes. Among these proteins, 7 have been reported to be able to regulate neurogenesis, while others may have the potential to regulate neurogenesis. This study profiles the major proteins released by astrocytes, which play important roles in the modulation of neurogenesis.

Proteomic analysis of astrocytic secretion that regulates neurogenesis using quantitative amine-specific isobaric tagging

International Nuclear Information System (INIS)

Yan, Hu; Zhou, Wenhao; Wei, Liming; Zhong, Fan; Yang, Yi

2010-01-01

Astrocytes are essential components of neurogenic niches that affect neurogenesis through membrane association and/or the release of soluble factors. To identify factors released from astrocytes that could regulate neural stem cell differentiation and proliferation, we used mild oxygen-glucose deprivation (OGD) to inhibit the secretory capacity of astrocytes. Using the Transwell co-culture system, we found that OGD-treated astrocytes could not promote neural stem cell differentiation and proliferation. Next, isobaric tagging for the relative and absolute quantitation (iTRAQ) proteomics techniques was performed to identify the proteins in the supernatants of astrocytes (with or without OGD). Through a multi-step analysis and gene ontology classification, 130 extracellular proteins were identified, most of which were involved in neuronal development, the inflammatory response, extracellular matrix composition and supportive functions. Of these proteins, 44 had never been reported to be produced by astrocytes. Using ProteinPilot software analysis, we found that 60 extracellular proteins were significantly altered (27 upregulated and 33 downregulated) in the supernatant of OGD-treated astrocytes. Among these proteins, 7 have been reported to be able to regulate neurogenesis, while others may have the potential to regulate neurogenesis. This study profiles the major proteins released by astrocytes, which play important roles in the modulation of neurogenesis.

Proteomics-based network analysis characterizes biological processes and pathways activated by preconditioned mesenchymal stem cells in cardiac repair mechanisms.

Science.gov (United States)

Di Silvestre, Dario; Brambilla, Francesca; Scardoni, Giovanni; Brunetti, Pietro; Motta, Sara; Matteucci, Marco; Laudanna, Carlo; Recchia, Fabio A; Lionetti, Vincenzo; Mauri, Pierluigi

2017-05-01

We have demonstrated that intramyocardial delivery of human mesenchymal stem cells preconditioned with a hyaluronan mixed ester of butyric and retinoic acid (MSCp + ) is more effective in preventing the decay of regional myocardial contractility in a swine model of myocardial infarction (MI). However, the understanding of the role of MSCp + in proteomic remodeling of cardiac infarcted tissue is not complete. We therefore sought to perform a comprehensive analysis of the proteome of infarct remote (RZ) and border zone (BZ) of pigs treated with MSCp + or unconditioned stem cells. Heart tissues were analyzed by MudPIT and differentially expressed proteins were selected by a label-free approach based on spectral counting. Protein profiles were evaluated by using PPI networks and their topological analysis. The proteomic remodeling was largely prevented in MSCp + group. Extracellular proteins involved in fibrosis were down-regulated, while energetic pathways were globally up-regulated. Cardioprotectant pathways involved in the production of keto acid metabolites were also activated. Additionally, we found that new hub proteins support the cardioprotective phenotype characterizing the left ventricular BZ treated with MSCp + . In fact, the up-regulation of angiogenic proteins NCL and RAC1 can be explained by the increase of capillary density induced by MSCp + . Our results show that angiogenic pathways appear to be uniquely positioned to integrate signaling with energetic pathways involving cardiac repair. Our findings prompt the use of proteomics-based network analysis to optimize new approaches preventing the post-ischemic proteomic remodeling that may underlie the limited self-repair ability of adult heart. Copyright © 2017 Elsevier B.V. All rights reserved.

[Methods of quantitative proteomics].

Science.gov (United States)

Kopylov, A T; Zgoda, V G

2007-01-01

In modern science proteomic analysis is inseparable from other fields of systemic biology. Possessing huge resources quantitative proteomics operates colossal information on molecular mechanisms of life. Advances in proteomics help researchers to solve complex problems of cell signaling, posttranslational modification, structure and functional homology of proteins, molecular diagnostics etc. More than 40 various methods have been developed in proteomics for quantitative analysis of proteins. Although each method is unique and has certain advantages and disadvantages all these use various isotope labels (tags). In this review we will consider the most popular and effective methods employing both chemical modifications of proteins and also metabolic and enzymatic methods of isotope labeling.

Impact of pyrrolidine-bispyrrole DNA minor groove binding agents and chirality on global proteomic profile in Escherichia Coli.

Science.gov (United States)

Yang, Ya-Ting; Lin, Chun-Yu; Jeng, Jingyueh; Ong, Chi-Wi

2013-05-23

There is great interest in the design of small molecules that selectively target minor grooves of duplex DNA for controlling specific gene expression implicated in a disease. The design of chiral small molecules for rational drug design has attracted increasing attention due to the chirality of DNA. Yet, there is limited research on the chirality effect of minor groove binders on DNA interaction, especially at the protein expression level. This paper is an attempt to illustrate that DNA binding affinity might not provide a full picture on the biological activities. Drug interacting at the genomic level can be translated to the proteomic level. Here we have illustrated that although the chiral bispyrrole-pyrrolidine-oligoamides, PySSPy and PyRSPy, showed low binding affinity to DNA, their influence at the proteomic level is significant. More importantly, the chirality also plays a role. Two-dimensional proteomic profile to identify the differentially expressed protein in Escherichia coli DH5α (E coli DH5α) were investigated. E coli DH5α incubated with the chiral PySSPy and PyRSPy, diastereomeric at the pyrrolidine ring, showed differential expression of eighteen proteins as observed through two dimensional proteomic profiling. These eighteen proteins identified by MALDI_TOF/TOF MS include antioxidant defense, DNA protection, protein synthesis, chaperone, and stress response proteins. No statistically significant toxicity was observed at the tested drug concentrations as measured via MTT assay. The current results showed that the chiral PySSPy and PyRSPy impact on the proteomic profiling of E coli DH5α, implicating the importance of drug chirality on biological activities at the molecular level.

Finding a Leucine in a Haystack: Searching the Proteome for ambigous Leucine-Aspartic Acid motifs

KAUST Repository

Arold, Stefan T.

2016-01-25

Leucine-aspartic acid (LD) motifs are short helical protein-protein interaction motifs involved in cell motility, survival and communication. LD motif interactions are also implicated in cancer metastasis and are targeted by several viruses. LD motifs are notoriously difficult to detect because sequence pattern searches lead to an excessively high number of false positives. Hence, despite 20 years of research, only six LD motif–containing proteins are known in humans, three of which are close homologues of the paxillin family. To enable the proteome-wide discovery of LD motifs, we developed LD Motif Finder (LDMF), a web tool based on machine learning that combines sequence information with structural predictions to detect LD motifs with high accuracy. LDMF predicted 13 new LD motifs in humans. Using biophysical assays, we experimentally confirmed in vitro interactions for four novel LD motif proteins. Thus, LDMF allows proteome-wide discovery of LD motifs, despite a highly ambiguous sequence pattern. Functional implications will be discussed.

Translational plant proteomics: A perspective

NARCIS (Netherlands)

Agrawal, G.K.; Pedreschi, R.; Barkla, B.J.; Bindschedler, L.V.; Cramer, R.; Sarkar, A.; Renaut, J.; Job, D.; Rakwal, R.

2012-01-01

Translational proteomics is an emerging sub-discipline of the proteomics field in the biological sciences. Translational plant proteomics aims to integrate knowledge from basic sciences to translate it into field applications to solve issues related but not limited to the recreational and economic

Evolution of Clinical Proteomics and its Role in Medicine | Office of Cancer Clinical Proteomics Research

Science.gov (United States)

NCI's Office of Cancer Clinical Proteomics Research authored a review of the current state of clinical proteomics in the peer-reviewed Journal of Proteome Research. The review highlights outcomes from the CPTC program and also provides a thorough overview of the different technologies that have pushed the field forward. Additionally, the review provides a vision for moving the field forward through linking advances in genomic and proteomic analysis to develop new, molecularly targeted interventions.

Proteomics of apheresis platelet supernatants during routine storage: Gender-related differences.

Science.gov (United States)

Dzieciatkowska, Monika; D'Alessandro, Angelo; Burke, Timothy A; Kelher, Marguerite R; Moore, Ernest E; Banerjee, Anirban; Silliman, Christopher C; West, Bernadette F; Hansen, Kirk C

2015-01-01

Proteomics has identified potential pathways involved in platelet storage lesions, which correlate with untoward effects in the recipient, including febrile non-haemolytic reactions. We hypothesize that an additional pathway involves protein mediators that accumulate in the platelet supernatants during routine storage in a donor gender-specific fashion. Apheresis platelet concentrates were collected from 5 healthy males and 5 females and routinely stored. The 14 most abundant plasma proteins were removed and the supernatant proteins from days 1 and 5 were analyzed via 1D-SDS-PAGE/nanoLC-MS/MS, before label-free quantitative proteomics analyses. Findings from a subset of 18 proteins were validated via LC-SRM analyses against stable isotope labeled standards. A total of 503 distinct proteins were detected in the platelet supernatants from the 4 sample groups: female or male donor platelets, either at storage day 1 or 5. Proteomics suggested a storage and gender-dependent impairment of blood coagulation mediators, pro-inflammatory complement components and cytokines, energy and redox metabolic enzymes. The supernatants from female donors demonstrated increased deregulation of structural proteins, extracellular matrix proteins and focal adhesion proteins, possibly indicating storage-dependent platelet activation. Routine storage of platelet concentrates induces changes in the supernatant proteome, which may have effects on the transfused patient, some of which are related to donor gender. The rationale behind this study is that protein components in platelet releasates have been increasingly observed to play a key role in adverse events and impaired homeostasis in transfused recipients. In this view, proteomics has recently emerged as a functional tool to address the issue of protein composition of platelet releasates from buffy coat-derived platelet concentrates in the blood bank. Despite early encouraging studies on buffy coat-derived platelet concentrates, platelet

Proteomic analysis of isolated chlamydomonas centrioles reveals orthologs of ciliary-disease genes.

Science.gov (United States)

Keller, Lani C; Romijn, Edwin P; Zamora, Ivan; Yates, John R; Marshall, Wallace F

2005-06-21

The centriole is one of the most enigmatic organelles in the cell. Centrioles are cylindrical, microtubule-based barrels found in the core of the centrosome. Centrioles also act as basal bodies during interphase to nucleate the assembly of cilia and flagella. There are currently only a handful of known centriole proteins. We used mass-spectrometry-based MudPIT (multidimensional protein identification technology) to identify the protein composition of basal bodies (centrioles) isolated from the green alga Chlamydomonas reinhardtii. This analysis detected the majority of known centriole proteins, including centrin, epsilon tubulin, and the cartwheel protein BLD10p. By combining proteomic data with information about gene expression and comparative genomics, we identified 45 cross-validated centriole candidate proteins in two classes. Members of the first class of proteins (BUG1-BUG27) are encoded by genes whose expression correlates with flagellar assembly and which therefore may play a role in ciliogenesis-related functions of basal bodies. Members of the second class (POC1-POC18) are implicated by comparative-genomics and -proteomics studies to be conserved components of the centriole. We confirmed centriolar localization for the human homologs of four candidate proteins. Three of the cross-validated centriole candidate proteins are encoded by orthologs of genes (OFD1, NPHP-4, and PACRG) implicated in mammalian ciliary function and disease, suggesting that oral-facial-digital syndrome and nephronophthisis may involve a dysfunction of centrioles and/or basal bodies. By analyzing isolated Chlamydomonas basal bodies, we have been able to obtain the first reported proteomic analysis of the centriole.

Plasma biomarker discovery in preeclampsia using a novel differential isolation technology for circulating extracellular vesicles.

Science.gov (United States)

Tan, Kok Hian; Tan, Soon Sim; Sze, Siu Kwan; Lee, Wai Kheong Ryan; Ng, Mor Jack; Lim, Sai Kiang

2014-10-01

To circumvent the complex protein milieu of plasma and discover robust predictive biomarkers for preeclampsia (PE), we investigate if phospholipid-binding ligands can reduce the milieu complexity by extracting plasma extracellular vesicles for biomarker discovery. Cholera toxin B chain (CTB) and annexin V (AV) which respectively binds GM1 ganglioside and phosphatidylserine were used to isolate extracellular vesicles from plasma of PE patients and healthy pregnant women. The proteins in the vesicles were identified using enzyme-linked immunosorbent assay, antibody array, and mass spectrometry. CTB and AV were found to bind 2 distinct groups of extracellular vesicles. Antibody array and enzyme-linked immunosorbent assay revealed that PE patients had elevated levels of CD105, interleukin-6, placental growth factor, tissue inhibitor of metallopeptidase 1, and atrial natriuretic peptide in cholera toxin B- but not AV-vesicles, and elevated levels of plasminogen activator inhibitor-1, pro-calcitonin, S100b, tumor growth factor β, vascular endothelial growth factor receptor 1, brain natriuretic peptide, and placental growth factor in both cholera toxin B- and AV-vesicles. CD9 level was elevated in cholera toxin B-vesicles but reduced in AV vesicles of PE patients. Proteome analysis revealed that in cholera toxin B-vesicles, 87 and 222 proteins were present only in PE patients and healthy pregnant women respectively while in AV-vesicles, 104 and 157 proteins were present only in PE and healthy pregnant women, respectively. This study demonstrated for the first time that CTB and AV bind unique extracellular vesicles, and their protein cargo reflects the disease state of the patient. The successful use of these 2 ligands to isolate circulating plasma extracellular vesicles for biomarker discovery in PE represents a novel technology for biomarker discovery that can be applied to other specialties. Copyright © 2014 Elsevier Inc. All rights reserved.

Proteomics of Maize Root Development.

Science.gov (United States)

Hochholdinger, Frank; Marcon, Caroline; Baldauf, Jutta A; Yu, Peng; Frey, Felix P

2018-01-01

Maize forms a complex root system with structurally and functionally diverse root types that are formed at different developmental stages to extract water and mineral nutrients from soil. In recent years proteomics has been intensively applied to identify proteins involved in shaping the three-dimensional architecture and regulating the function of the maize root system. With the help of developmental mutants, proteomic changes during the initiation and emergence of shoot-borne, lateral and seminal roots have been examined. Furthermore, root hairs were surveyed to understand the proteomic changes during the elongation of these single cell type structures. In addition, primary roots have been used to study developmental changes of the proteome but also to investigate the proteomes of distinct tissues such as the meristematic zone, the elongation zone as well as stele and cortex of the differentiation zone. Moreover, subcellular fractions of the primary root including cell walls, plasma membranes and secreted mucilage have been analyzed. Finally, the superior vigor of hybrid seedling roots compared to their parental inbred lines was studied on the proteome level. In summary, these studies provide novel insights into the complex proteomic interactions of the elaborate maize root system during development.

Proteomics of Maize Root Development

Directory of Open Access Journals (Sweden)

Frank Hochholdinger

2018-03-01

Full Text Available Maize forms a complex root system with structurally and functionally diverse root types that are formed at different developmental stages to extract water and mineral nutrients from soil. In recent years proteomics has been intensively applied to identify proteins involved in shaping the three-dimensional architecture and regulating the function of the maize root system. With the help of developmental mutants, proteomic changes during the initiation and emergence of shoot-borne, lateral and seminal roots have been examined. Furthermore, root hairs were surveyed to understand the proteomic changes during the elongation of these single cell type structures. In addition, primary roots have been used to study developmental changes of the proteome but also to investigate the proteomes of distinct tissues such as the meristematic zone, the elongation zone as well as stele and cortex of the differentiation zone. Moreover, subcellular fractions of the primary root including cell walls, plasma membranes and secreted mucilage have been analyzed. Finally, the superior vigor of hybrid seedling roots compared to their parental inbred lines was studied on the proteome level. In summary, these studies provide novel insights into the complex proteomic interactions of the elaborate maize root system during development.

Integrating cell biology and proteomic approaches in plants.

Science.gov (United States)

Takáč, Tomáš; Šamajová, Olga; Šamaj, Jozef

2017-10-03

Significant improvements of protein extraction, separation, mass spectrometry and bioinformatics nurtured advancements of proteomics during the past years. The usefulness of proteomics in the investigation of biological problems can be enhanced by integration with other experimental methods from cell biology, genetics, biochemistry, pharmacology, molecular biology and other omics approaches including transcriptomics and metabolomics. This review aims to summarize current trends integrating cell biology and proteomics in plant science. Cell biology approaches are most frequently used in proteomic studies investigating subcellular and developmental proteomes, however, they were also employed in proteomic studies exploring abiotic and biotic stress responses, vesicular transport, cytoskeleton and protein posttranslational modifications. They are used either for detailed cellular or ultrastructural characterization of the object subjected to proteomic study, validation of proteomic results or to expand proteomic data. In this respect, a broad spectrum of methods is employed to support proteomic studies including ultrastructural electron microscopy studies, histochemical staining, immunochemical localization, in vivo imaging of fluorescently tagged proteins and visualization of protein-protein interactions. Thus, cell biological observations on fixed or living cell compartments, cells, tissues and organs are feasible, and in some cases fundamental for the validation and complementation of proteomic data. Validation of proteomic data by independent experimental methods requires development of new complementary approaches. Benefits of cell biology methods and techniques are not sufficiently highlighted in current proteomic studies. This encouraged us to review most popular cell biology methods used in proteomic studies and to evaluate their relevance and potential for proteomic data validation and enrichment of purely proteomic analyses. We also provide examples of

Genomes to Proteomes

Energy Technology Data Exchange (ETDEWEB)

Panisko, Ellen A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Grigoriev, Igor [USDOE Joint Genome Inst., Walnut Creek, CA (United States); Daly, Don S. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Webb-Robertson, Bobbie-Jo [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Baker, Scott E. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

2009-03-01

Biologists are awash with genomic sequence data. In large part, this is due to the rapid acceleration in the generation of DNA sequence that occurred as public and private research institutes raced to sequence the human genome. In parallel with the large human genome effort, mostly smaller genomes of other important model organisms were sequenced. Projects following on these initial efforts have made use of technological advances and the DNA sequencing infrastructure that was built for the human and other organism genome projects. As a result, the genome sequences of many organisms are available in high quality draft form. While in many ways this is good news, there are limitations to the biological insights that can be gleaned from DNA sequences alone; genome sequences offer only a bird's eye view of the biological processes endemic to an organism or community. Fortunately, the genome sequences now being produced at such a high rate can serve as the foundation for other global experimental platforms such as proteomics. Proteomic methods offer a snapshot of the proteins present at a point in time for a given biological sample. Current global proteomics methods combine enzymatic digestion, separations, mass spectrometry and database searching for peptide identification. One key aspect of proteomics is the prediction of peptide sequences from mass spectrometry data. Global proteomic analysis uses computational matching of experimental mass spectra with predicted spectra based on databases of gene models that are often generated computationally. Thus, the quality of gene models predicted from a genome sequence is crucial in the generation of high quality peptide identifications. Once peptides are identified they can be assigned to their parent protein. Proteins identified as expressed in a given experiment are most useful when compared to other expressed proteins in a larger biological context or biochemical pathway. In this chapter we will discuss the automatic

In-depth Proteomics Characterization of Embryogenesis of the Honey Bee Worker (Apis mellifera ligustica) *

Science.gov (United States)

Fang, Yu; Feng, Mao; Han, Bin; Lu, Xiaoshan; Ramadan, Haitham; Li, Jianke

2014-01-01

Identifying proteome changes of honey bee embryogenesis is of prime importance for unraveling the molecular mechanisms that they underlie. However, many proteomic changes during the embryonic period are not well characterized. We analyzed the proteomic alterations over the complete time course of honey bee worker embryogenesis at 24, 48, and 72 h of age, using mass spectrometry-based proteomics, label-free quantitation, and bioinformatics. Of the 1460 proteins identified the embryo of all three ages, the core proteome (proteins shared by the embryos of all three ages, accounting for 40%) was mainly involved in protein synthesis, metabolic energy, development, and molecular transporter, which indicates their centrality in driving embryogenesis. However, embryos at different developmental stages have their own specific proteome and pathway signatures to coordinate and modulate developmental events. The young embryos (proteins related to nutrition storage and nucleic acid metabolism may correlate with the cell proliferation occurring at this stage. The middle aged embryos (24–48 h) enhanced expression of proteins associated with cell cycle control, transporters, antioxidant activity, and the cytoskeleton suggest their roles to support rudimentary organogenesis. Among these proteins, the biological pathways of aminoacyl-tRNA biosynthesis, β-alanine metabolism, and protein export are intensively activated in the embryos of middle age. The old embryos (48–72 h) elevated expression of proteins implicated in fatty acid metabolism and morphogenesis indicate their functionality for the formation and development of organs and dorsal closure, in which the biological pathways of fatty acid metabolism and RNA transport are highly activated. These findings add novel understanding to the molecular details of honey bee embryogenesis, in which the programmed activation of the proteome matches with the physiological transition observed during embryogenesis. The identified

The role of Aspergillus flavus veA in the production of extracellular proteins during growth on starch substrates.

Science.gov (United States)

Duran, Rocio M; Gregersen, Scott; Smith, Timothy D; Bhetariya, Preetida J; Cary, Jeffrey W; Harris-Coward, Pamela Y; Mattison, Christopher P; Grimm, Casey; Calvo, Ana M

2014-06-01

The aflatoxin-producer and opportunistic plant pathogenic, filamentous fungus Aspergillus flavus is responsible for the contamination of corn and other important agricultural commodities. In order to obtain nutrients from the host A. flavus produces a variety of extracellular hydrolytic enzymes. Interestingly, A. flavus amylase and protease activity are dependent on the global regulator veA, a gene known to regulate morphogenesis and secondary metabolism in numerous fungi. Analysis of starch degradation by fungal enzymes secreted into broths of starch- or corn kernel-based media showed a notable accumulation of glucose in samples of the A. flavus control strain while the deletion veA sample accumulated high levels of maltose and maltotriose and only a small amount of glucose. Furthermore, SDS-PAGE and proteomics analysis of culture broths from starch- or corn kernel-based media demonstrated differential production of a number of proteins that included a reduction in the amount of a glucoamylase protein in the veA mutant compared to the control strain, while an alpha-amylase was produced in greater quantities in the veA mutant. Quantitative real-time PCR and western blot analyses using anti-glucoamylase or alpha-amylase antisera supported the proteomics results. Additionally, an overall reduction in protease activity was observed in the veA mutant including production of the alkaline protease, oryzin, compared to the control strain. These findings contribute to our knowledge of mechanisms controlling production of hydrolases and other extracellular proteins during growth of A. flavus on natural starch-based substrates.

EcmPred: Prediction of extracellular matrix proteins based on random forest with maximum relevance minimum redundancy feature selection

KAUST Repository

Kandaswamy, Krishna Kumar Umar

2013-01-01

The extracellular matrix (ECM) is a major component of tissues of multicellular organisms. It consists of secreted macromolecules, mainly polysaccharides and glycoproteins. Malfunctions of ECM proteins lead to severe disorders such as marfan syndrome, osteogenesis imperfecta, numerous chondrodysplasias, and skin diseases. In this work, we report a random forest approach, EcmPred, for the prediction of ECM proteins from protein sequences. EcmPred was trained on a dataset containing 300 ECM and 300 non-ECM and tested on a dataset containing 145 ECM and 4187 non-ECM proteins. EcmPred achieved 83% accuracy on the training and 77% on the test dataset. EcmPred predicted 15 out of 20 experimentally verified ECM proteins. By scanning the entire human proteome, we predicted novel ECM proteins validated with gene ontology and InterPro. The dataset and standalone version of the EcmPred software is available at http://www.inb.uni-luebeck.de/tools-demos/Extracellular_matrix_proteins/EcmPred. © 2012 Elsevier Ltd.

Proteomic explorations of autism spectrum disorder.

Science.gov (United States)

Szoko, Nicholas; McShane, Adam J; Natowicz, Marvin R

2017-09-01

Proteomics, the large-scale study of protein expression in cells and tissues, is a powerful tool to study the biology of clinical conditions and has provided significant insights in many experimental systems. Herein, we review the basics of proteomic methodology and discuss challenges in using proteomic approaches to study autism. Unlike other experimental approaches, such as genomic approaches, there have been few large-scale studies of proteins in tissues from persons with autism. Most of the proteomic studies on autism used blood or other peripheral tissues; few studies used brain tissue. Some studies found dysregulation of aspects of the immune system or of aspects of lipid metabolism, but no consistent findings were noted. Based on the challenges in using proteomics to study autism, we discuss considerations for future studies. Apart from the complex technical considerations implicit in any proteomic analysis, key nontechnical matters include attention to subject and specimen inclusion/exclusion criteria, having adequate sample size to ensure appropriate powering of the study, attention to the state of specimens prior to proteomic analysis, and the use of a replicate set of specimens, when possible. We conclude by discussing some potentially productive uses of proteomics, potentially coupled with other approaches, for future autism research including: (1) proteomic analysis of banked human brain specimens; (2) proteomic analysis of tissues from animal models of autism; and (3) proteomic analysis of induced pluripotent stem cells that are differentiated into various types of brain cells and neural organoids. Autism Res 2017, 10: 1460-1469. © 2017 International Society for Autism Research, Wiley Periodicals, Inc. © 2017 International Society for Autism Research, Wiley Periodicals, Inc.

Proteomics dataset

DEFF Research Database (Denmark)

Bennike, Tue Bjerg; Carlsen, Thomas Gelsing; Ellingsen, Torkell

2017-01-01

patients (Morgan et al., 2012; Abraham and Medzhitov, 2011; Bennike, 2014) [8–10. Therefore, we characterized the proteome of colon mucosa biopsies from 10 inflammatory bowel disease ulcerative colitis (UC) patients, 11 gastrointestinal healthy rheumatoid arthritis (RA) patients, and 10 controls. We...... been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD001608 for ulcerative colitis and control samples, and PXD003082 for rheumatoid arthritis samples....

Proteome alteration induced by hTERT transfection of human fibroblast cells.

Science.gov (United States)

Mazzucchelli, Gabriel D; Gabelica, Valérie; Smargiasso, Nicolas; Fléron, Maximilien; Ashimwe, Wilson; Rosu, Frédéric; De Pauw-Gillet, Marie-Claire; Riou, Jean-François; De Pauw, Edwin

2008-04-17

Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human telomerase reverse transcriptase (hTERT) gene. hTERT transfected cell lines may be of outstanding interest to monitor the effect of drugs targeting the telomerase activity. The incidence of hTERT gene transfection at the proteome level is a prerequisite to that purpose. The effect of the transfection has been studied on the proteome of human fibroblast (WI38). Cytosolic and nuclear fractions of WI38 cells, empty vector transfected WI38 (WI38-HPV) and hTERT WI38 cells were submitted to a 2D-DIGE (Two-Dimensional Differential In-Gel Electrophoresis) analysis. Only spots that had a similar abundance in WI38 and WI38-HPV, but were differentially expressed in WI38 hTERT were selected for MS identification. This method directly points to the proteins linked with the hTERT expression. Number of false positive differentially expressed proteins has been excluded by using control WI38-HPV cells. The proteome alteration induced by hTERT WI38 transfection should be taken into account in subsequent use of the cell line for anti-telomerase drugs evaluation. 2D-DIGE experiment shows that 57 spots out of 2246 are significantly differentially expressed in the cytosolic fraction due to hTERT transfection, and 38 were confidently identified. In the nuclear fraction, 44 spots out of 2172 were selected in the differential proteome analysis, and 14 were identified. The results show that, in addition to elongating telomeres, hTERT gene transfection has other physiological roles, among which an enhanced ER capacity and a potent cell protection against apoptosis. We show that the methodology reduces the complexity of the proteome analysis and highlights proteins implicated in other processes than telomere elongation. hTERT induced proteome changes suggest that telomerase expression enhances natural cell repair

Proteome alteration induced by hTERT transfection of human fibroblast cells

Directory of Open Access Journals (Sweden)

Riou Jean-François

2008-04-01

Full Text Available Abstract Background Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human telomerase reverse transcriptase (hTERT gene. hTERT transfected cell lines may be of outstanding interest to monitor the effect of drugs targeting the telomerase activity. The incidence of hTERT gene transfection at the proteome level is a prerequisite to that purpose. The effect of the transfection has been studied on the proteome of human fibroblast (WI38. Cytosolic and nuclear fractions of WI38 cells, empty vector transfected WI38 (WI38-HPV and hTERT WI38 cells were submitted to a 2D-DIGE (Two-Dimensional Differential In-Gel Electrophoresis analysis. Only spots that had a similar abundance in WI38 and WI38-HPV, but were differentially expressed in WI38 hTERT were selected for MS identification. This method directly points to the proteins linked with the hTERT expression. Number of false positive differentially expressed proteins has been excluded by using control WI38-HPV cells. The proteome alteration induced by hTERT WI38 transfection should be taken into account in subsequent use of the cell line for anti-telomerase drugs evaluation. Results 2D-DIGE experiment shows that 57 spots out of 2246 are significantly differentially expressed in the cytosolic fraction due to hTERT transfection, and 38 were confidently identified. In the nuclear fraction, 44 spots out of 2172 were selected in the differential proteome analysis, and 14 were identified. The results show that, in addition to elongating telomeres, hTERT gene transfection has other physiological roles, among which an enhanced ER capacity and a potent cell protection against apoptosis. Conclusion We show that the methodology reduces the complexity of the proteome analysis and highlights proteins implicated in other processes than telomere elongation. hTERT induced proteome changes suggest

Neuropeptide Y infusion into the shell region of the rat nucleus accumbens increases extracellular levels of dopamine

DEFF Research Database (Denmark)

Sørensen, Gunnar; Wegener, Gregers; Hasselstrøm, Jørgen

2009-01-01

Increases in extracellular dopamine in the shell region of the nucleus accumbens are centrally involved in mediating reinforcement of addictive drugs. Neuropeptide Y (NPY) and its receptors are present in the nucleus accumbens and have been implicated in addiction mechanisms. This study further...... explored the potential role of NPY in addiction mechanisms using microdialysis to measure extracellular dopamine in vivo after infusion of NPY directly into the accumbal shell region of adult rats. NPY was found to dose-dependently increase extracellular dopamine levels, indicating that NPY could play...... an important role in drug reinforcement by modulating accumbal dopamine levels...

Proteomic and functional profiles of a follicle-stimulating hormone positive human nonfunctional pituitary adenoma.

Science.gov (United States)

Wang, Xiaowei; Guo, Tianyao; Peng, Fang; Long, Ying; Mu, Yun; Yang, Haiyan; Ye, Ningrong; Li, Xuejun; Zhan, Xianquan

2015-06-01

Nonfunctional pituitary adenoma (NFPA) is highly heterogeneous with different hormone-expressed subtypes in NFPA tissues including follicle-stimulating hormone (FSH) positive, luteinizing hormone-positive, FSH/luteinizing hormone-positive, and negative types. To analyze in-depth the variations in the proteomes among different NFPA subtypes for our long-term goal to clarify molecular mechanisms of NFPA and to detect tumor biomarker for personalized medicine practice, a reference map of proteome of a human FSH-expressed NFPA tissue was described here. 2DE and PDQuest image analysis were used to array each protein. MALDI-TOF PMF and human Swiss-Prot databases with MASCOT search were used to identify each protein. A good 2DE pattern with high level of between-gel reproducibility was attained with an average positional deviation 1.98 ± 0.75 mm in the IEF direction and 1.62 ± 0.68 mm in the SDS-PAGE direction. Approximately 1200 protein spots were 2DE-detected and 192 redundant proteins that were contained in 141 protein spots were PMF-identified, representing 107 nonredundant proteins. Those proteins were located in cytoplasm, nucleus, plasma membrane, extracellular space, and so on, and those functioned in transmembrane receptor, ion channel, transcription/translation regulator, transporter, enzyme, phosphatase, kinase, and so on. Several important pathway networks were characterized from those identified proteins with DAVID and Ingenuity Pathway Analysis systems, including gluconeogenesis and glycolysis, mitochondrial dysfunction, oxidative stress, cell-cycle alteration, MAPKsignaling system, immune response, TP53-signaling, VEGF-signaling, and inflammation signaling pathways. Those resulting data contribute to a functional profile of the proteome of a human FSH-positive NFPA tissue, and will serve as a reference for the heterogeneity analysis of NFPA proteomes. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

1001 Proteomes: a functional proteomics portal for the analysis of Arabidopsis thaliana accessions.

Science.gov (United States)

Joshi, Hiren J; Christiansen, Katy M; Fitz, Joffrey; Cao, Jun; Lipzen, Anna; Martin, Joel; Smith-Moritz, A Michelle; Pennacchio, Len A; Schackwitz, Wendy S; Weigel, Detlef; Heazlewood, Joshua L

2012-05-15

The sequencing of over a thousand natural strains of the model plant Arabidopsis thaliana is producing unparalleled information at the genetic level for plant researchers. To enable the rapid exploitation of these data for functional proteomics studies, we have created a resource for the visualization of protein information and proteomic datasets for sequenced natural strains of A. thaliana. The 1001 Proteomes portal can be used to visualize amino acid substitutions or non-synonymous single-nucleotide polymorphisms in individual proteins of A. thaliana based on the reference genome Col-0. We have used the available processed sequence information to analyze the conservation of known residues subject to protein phosphorylation among these natural strains. The substitution of amino acids in A. thaliana natural strains is heavily constrained and is likely a result of the conservation of functional attributes within proteins. At a practical level, we demonstrate that this information can be used to clarify ambiguously defined phosphorylation sites from phosphoproteomic studies. Protein sets of available natural variants are available for download to enable proteomic studies on these accessions. Together this information can be used to uncover the possible roles of specific amino acids in determining the structure and function of proteins in the model plant A. thaliana. An online portal to enable the community to exploit these data can be accessed at http://1001proteomes.masc-proteomics.org/

Xylem sap proteomics.

Science.gov (United States)

de Bernonville, Thomas Dugé; Albenne, Cécile; Arlat, Matthieu; Hoffmann, Laurent; Lauber, Emmanuelle; Jamet, Elisabeth

2014-01-01

Proteomic analysis of xylem sap has recently become a major field of interest to understand several biological questions related to plant development and responses to environmental clues. The xylem sap appears as a dynamic fluid undergoing changes in its proteome upon abiotic and biotic stresses. Unlike cell compartments which are amenable to purification in sufficient amount prior to proteomic analysis, the xylem sap has to be collected in particular conditions to avoid contamination by intracellular proteins and to obtain enough material. A model plant like Arabidopsis thaliana is not suitable for such an analysis because efficient harvesting of xylem sap is difficult. The analysis of the xylem sap proteome also requires specific procedures to concentrate proteins and to focus on proteins predicted to be secreted. Indeed, xylem sap proteins appear to be synthesized and secreted in the root stele or to originate from dying differentiated xylem cells. This chapter describes protocols to collect xylem sap from Brassica species and to prepare total and N-glycoprotein extracts for identification of proteins by mass spectrometry analyses and bioinformatics.

Farm animal proteomics - A review

DEFF Research Database (Denmark)

Bendixen, Emøke; Danielsen, Marianne; Hollung, Kristin

2011-01-01

In agricultural sciences as in all other areas of life science, the implementation of proteomics and other post-genomic tools is an important step towards more detailed understanding of the complex biological systems that control physiology and pathology of living beings. Farm animals are raised...... and cattle are relevant not only for farm animal sciences, but also for adding to our understanding of complex biological mechanisms of health and disease in humans. The aim of this review is to present an overview of the specific topics of interest within farm animal proteomics, and to highlight some...... of the areas where synergy between classic model organism proteomics and farm animal proteomics is rapidly emerging. Focus will be on introducing the special biological traits that play an important role in food production, and on how proteomics may help optimize farm animal production...

Proteomic analysis of human tooth pulp: proteomics of human tooth.

Science.gov (United States)

Eckhardt, Adam; Jágr, Michal; Pataridis, Statis; Mikšík, Ivan

2014-12-01

The unique pulp-dentin complex demonstrates strong regenerative potential, which enables it to respond to disease and traumatic injury. Identifying the proteins of the pulp-dentin complex is crucial to understanding the mechanisms of regeneration, tissue calcification, defense processes, and the reparation of dentin by dental pulp. The lack of knowledge of these proteins limits the development of more efficient therapies. The proteomic profile of human tooth pulp was investigated and compared with the proteome of human dentin and blood. The samples of tooth pulp were obtained from 5 sound permanent human third molars of 5 adults (n = 5). The extracted proteins were separated by 2-dimensional gel electrophoresis, analyzed by nano-liquid chromatography tandem mass spectrometry, and identified by correlating mass spectra to the proteomic databases. A total of 342 proteins were identified with high confidence, and 2 proteins were detected for the first time in an actual human sample. The identified tooth pulp proteins have a variety of functions: structural, catalytic, transporter, protease activity, immune response, and many others. In a comparison with dentin and blood plasma, 140 (pulp/dentin) shared proteins were identified, 37 of which were not observed in plasma. It can be suggested that they might participate in the unique pulp-dentin complex. This proteomic investigation of human tooth pulp, together with the previously published study of human dentin, is one of the most comprehensive proteome lists of human teeth to date. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Mining the granule proteome

DEFF Research Database (Denmark)

Albrethsen, Jakob; Goetze, Jens P; Johnsen, Anders H

2015-01-01

Proteomics of secretory granules is an emerging strategy for identifying secreted proteins, including potentially novel candidate biomarkers and peptide hormones. In addition, proteomics can provide information about the abundance, localization and structure (post-translational modification) of g...

Advances of Proteomic Sciences in Dentistry.

Science.gov (United States)

Khurshid, Zohaib; Zohaib, Sana; Najeeb, Shariq; Zafar, Muhammad Sohail; Rehman, Rabia; Rehman, Ihtesham Ur

2016-05-13

Applications of proteomics tools revolutionized various biomedical disciplines such as genetics, molecular biology, medicine, and dentistry. The aim of this review is to highlight the major milestones in proteomics in dentistry during the last fifteen years. Human oral cavity contains hard and soft tissues and various biofluids including saliva and crevicular fluid. Proteomics has brought revolution in dentistry by helping in the early diagnosis of various diseases identified by the detection of numerous biomarkers present in the oral fluids. This paper covers the role of proteomics tools for the analysis of oral tissues. In addition, dental materials proteomics and their future directions are discussed.

Proteomics - new analytical approaches

International Nuclear Information System (INIS)

Hancock, W.S.

2001-01-01

Full text: Recent developments in the sequencing of the human genome have indicated that the number of coding gene sequences may be as few as 30,000. It is clear, however, that the complexity of the human species is dependent on the much greater diversity of the corresponding protein complement. Estimates of the diversity (discrete protein species) of the human proteome range from 200,000 to 300,000 at the lower end to 2,000,000 to 3,000,000 at the high end. In addition, proteomics (the study of the protein complement to the genome) has been subdivided into two main approaches. Global proteomics refers to a high throughput examination of the full protein set present in a cell under a given environmental condition. Focused proteomics refers to a more detailed study of a restricted set of proteins that are related to a specified biochemical pathway or subcellular structure. While many of the advances in proteomics will be based on the sequencing of the human genome, de novo characterization of protein microheterogeneity (glycosylation, phosphorylation and sulfation as well as the incorporation of lipid components) will be required in disease studies. To characterize these modifications it is necessary to digest the protein mixture with an enzyme to produce the corresponding mixture of peptides. In a process analogous to sequencing of the genome, shot-gun sequencing of the proteome is based on the characterization of the key fragments produced by such a digest. Thus, a glycopeptide and hence a specific glycosylation motif will be identified by a unique mass and then a diagnostic MS/MS spectrum. Mass spectrometry will be the preferred detector in these applications because of the unparalleled information content provided by one or more dimensions of mass measurement. In addition, highly efficient separation processes are an absolute requirement for advanced proteomic studies. For example, a combination of the orthogonal approaches, HPLC and HPCE, can be very powerful

Integration of gel-based and gel-free proteomic data for functional analysis of proteins through Soybean Proteome Database

KAUST Repository

Komatsu, Setsuko

2017-05-10

The Soybean Proteome Database (SPD) stores data on soybean proteins obtained with gel-based and gel-free proteomic techniques. The database was constructed to provide information on proteins for functional analyses. The majority of the data is focused on soybean (Glycine max ‘Enrei’). The growth and yield of soybean are strongly affected by environmental stresses such as flooding. The database was originally constructed using data on soybean proteins separated by two-dimensional polyacrylamide gel electrophoresis, which is a gel-based proteomic technique. Since 2015, the database has been expanded to incorporate data obtained by label-free mass spectrometry-based quantitative proteomics, which is a gel-free proteomic technique. Here, the portions of the database consisting of gel-free proteomic data are described. The gel-free proteomic database contains 39,212 proteins identified in 63 sample sets, such as temporal and organ-specific samples of soybean plants grown under flooding stress or non-stressed conditions. In addition, data on organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored. Furthermore, the database integrates multiple omics data such as genomics, transcriptomics, metabolomics, and proteomics. The SPD database is accessible at http://proteome.dc.affrc.go.jp/Soybean/. Biological significanceThe Soybean Proteome Database stores data obtained from both gel-based and gel-free proteomic techniques. The gel-free proteomic database comprises 39,212 proteins identified in 63 sample sets, such as different organs of soybean plants grown under flooding stress or non-stressed conditions in a time-dependent manner. In addition, organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored in the gel-free proteomics database. A total of 44,704 proteins, including 5490 proteins identified using a gel-based proteomic technique, are stored in the SPD. It accounts for approximately 80% of all

Integration of gel-based and gel-free proteomic data for functional analysis of proteins through Soybean Proteome Database.

Science.gov (United States)

Komatsu, Setsuko; Wang, Xin; Yin, Xiaojian; Nanjo, Yohei; Ohyanagi, Hajime; Sakata, Katsumi

2017-06-23

The Soybean Proteome Database (SPD) stores data on soybean proteins obtained with gel-based and gel-free proteomic techniques. The database was constructed to provide information on proteins for functional analyses. The majority of the data is focused on soybean (Glycine max 'Enrei'). The growth and yield of soybean are strongly affected by environmental stresses such as flooding. The database was originally constructed using data on soybean proteins separated by two-dimensional polyacrylamide gel electrophoresis, which is a gel-based proteomic technique. Since 2015, the database has been expanded to incorporate data obtained by label-free mass spectrometry-based quantitative proteomics, which is a gel-free proteomic technique. Here, the portions of the database consisting of gel-free proteomic data are described. The gel-free proteomic database contains 39,212 proteins identified in 63 sample sets, such as temporal and organ-specific samples of soybean plants grown under flooding stress or non-stressed conditions. In addition, data on organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored. Furthermore, the database integrates multiple omics data such as genomics, transcriptomics, metabolomics, and proteomics. The SPD database is accessible at http://proteome.dc.affrc.go.jp/Soybean/. The Soybean Proteome Database stores data obtained from both gel-based and gel-free proteomic techniques. The gel-free proteomic database comprises 39,212 proteins identified in 63 sample sets, such as different organs of soybean plants grown under flooding stress or non-stressed conditions in a time-dependent manner. In addition, organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored in the gel-free proteomics database. A total of 44,704 proteins, including 5490 proteins identified using a gel-based proteomic technique, are stored in the SPD. It accounts for approximately 80% of all predicted proteins from

Integration of gel-based and gel-free proteomic data for functional analysis of proteins through Soybean Proteome Database

KAUST Repository

Komatsu, Setsuko; Wang, Xin; Yin, Xiaojian; Nanjo, Yohei; Ohyanagi, Hajime; Sakata, Katsumi

2017-01-01

The Soybean Proteome Database (SPD) stores data on soybean proteins obtained with gel-based and gel-free proteomic techniques. The database was constructed to provide information on proteins for functional analyses. The majority of the data is focused on soybean (Glycine max ‘Enrei’). The growth and yield of soybean are strongly affected by environmental stresses such as flooding. The database was originally constructed using data on soybean proteins separated by two-dimensional polyacrylamide gel electrophoresis, which is a gel-based proteomic technique. Since 2015, the database has been expanded to incorporate data obtained by label-free mass spectrometry-based quantitative proteomics, which is a gel-free proteomic technique. Here, the portions of the database consisting of gel-free proteomic data are described. The gel-free proteomic database contains 39,212 proteins identified in 63 sample sets, such as temporal and organ-specific samples of soybean plants grown under flooding stress or non-stressed conditions. In addition, data on organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored. Furthermore, the database integrates multiple omics data such as genomics, transcriptomics, metabolomics, and proteomics. The SPD database is accessible at http://proteome.dc.affrc.go.jp/Soybean/. Biological significanceThe Soybean Proteome Database stores data obtained from both gel-based and gel-free proteomic techniques. The gel-free proteomic database comprises 39,212 proteins identified in 63 sample sets, such as different organs of soybean plants grown under flooding stress or non-stressed conditions in a time-dependent manner. In addition, organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored in the gel-free proteomics database. A total of 44,704 proteins, including 5490 proteins identified using a gel-based proteomic technique, are stored in the SPD. It accounts for approximately 80% of all

Elucidation of cross-species proteomic effects in human and hominin bone proteome identification through a bioinformatics experiment

DEFF Research Database (Denmark)

Welker, F.

2018-01-01

Background: The study of ancient protein sequences is increasingly focused on the analysis of older samples, including those of ancient hominins. The analysis of such ancient proteomes thereby potentially suffers from "cross-species proteomic effects": the loss of peptide and protein identificati......Background: The study of ancient protein sequences is increasingly focused on the analysis of older samples, including those of ancient hominins. The analysis of such ancient proteomes thereby potentially suffers from "cross-species proteomic effects": the loss of peptide and protein...... not been demonstrated. If error-tolerant searches do not overcome the cross-species proteomic issue then there might be inherent biases in the identified proteomes. Here, a bioinformatics experiment is performed to test this using a set of modern human bone proteomes and three independent searches against......), but roughly half of the mutable PSMs were not recovered. As a result, peptide and protein identification rates are higher in error-tolerant mode compared to non-error-tolerant searches but did not recover protein identifications completely. Data indicates that peptide length and the number of mutations...

Exploring the Arabidopsis Proteome: Influence of Protein Solubilization Buffers on Proteome Coverage

KAUST Repository

Marondedze, Claudius; Wong, Aloysius Tze; Groen, Arnoud; Serano, Natalia Lorena Gorron; Jankovic, Boris R.; Lilley, Kathryn; Gehring, Christoph A; Thomas, Ludivine

2014-01-01

The study of proteomes provides new insights into stimulus-specific responses of protein synthesis and turnover, and the role of post-translational modifications at the systems level. Due to the diverse chemical nature of proteins and shortcomings in the analytical techniques used in their study, only a partial display of the proteome is achieved in any study, and this holds particularly true for plant proteomes. Here we show that different solubilization and separation methods have profound effects on the resulting proteome. In particular, we observed that the type of detergents employed in the solubilization buffer preferentially enriches proteins in different functional categories. These include proteins with a role in signaling, transport, response to temperature stimuli and metabolism. This data may offer a functional bias on comparative analysis studies. In order to obtain a broader coverage, we propose a two-step solubilization protocol with first a detergent-free buffer and then a second step utilizing a combination of two detergents to solubilize proteins.

Exploring the Arabidopsis Proteome: Influence of Protein Solubilization Buffers on Proteome Coverage

Directory of Open Access Journals (Sweden)

Claudius Marondedze

2014-12-01

Full Text Available The study of proteomes provides new insights into stimulus-specific responses of protein synthesis and turnover, and the role of post-translational modifications at the systems level. Due to the diverse chemical nature of proteins and shortcomings in the analytical techniques used in their study, only a partial display of the proteome is achieved in any study, and this holds particularly true for plant proteomes. Here we show that different solubilization and separation methods have profound effects on the resulting proteome. In particular, we observed that the type of detergents employed in the solubilization buffer preferentially enriches proteins in different functional categories. These include proteins with a role in signaling, transport, response to temperature stimuli and metabolism. This data may offer a functional bias on comparative analysis studies. In order to obtain a broader coverage, we propose a two-step solubilization protocol with first a detergent-free buffer and then a second step utilizing a combination of two detergents to solubilize proteins.

Exploring the Arabidopsis Proteome: Influence of Protein Solubilization Buffers on Proteome Coverage

KAUST Repository

Marondedze, Claudius

2014-12-31

The study of proteomes provides new insights into stimulus-specific responses of protein synthesis and turnover, and the role of post-translational modifications at the systems level. Due to the diverse chemical nature of proteins and shortcomings in the analytical techniques used in their study, only a partial display of the proteome is achieved in any study, and this holds particularly true for plant proteomes. Here we show that different solubilization and separation methods have profound effects on the resulting proteome. In particular, we observed that the type of detergents employed in the solubilization buffer preferentially enriches proteins in different functional categories. These include proteins with a role in signaling, transport, response to temperature stimuli and metabolism. This data may offer a functional bias on comparative analysis studies. In order to obtain a broader coverage, we propose a two-step solubilization protocol with first a detergent-free buffer and then a second step utilizing a combination of two detergents to solubilize proteins.

Halobacterium salinarum NRC-1 PeptideAtlas: toward strategies for targeted proteomics and improved proteome coverage.

Science.gov (United States)

Van, Phu T; Schmid, Amy K; King, Nichole L; Kaur, Amardeep; Pan, Min; Whitehead, Kenia; Koide, Tie; Facciotti, Marc T; Goo, Young Ah; Deutsch, Eric W; Reiss, David J; Mallick, Parag; Baliga, Nitin S

2008-09-01

The relatively small numbers of proteins and fewer possible post-translational modifications in microbes provide a unique opportunity to comprehensively characterize their dynamic proteomes. We have constructed a PeptideAtlas (PA) covering 62.7% of the predicted proteome of the extremely halophilic archaeon Halobacterium salinarum NRC-1 by compiling approximately 636 000 tandem mass spectra from 497 mass spectrometry runs in 88 experiments. Analysis of the PA with respect to biophysical properties of constituent peptides, functional properties of parent proteins of detected peptides, and performance of different mass spectrometry approaches has highlighted plausible strategies for improving proteome coverage and selecting signature peptides for targeted proteomics. Notably, discovery of a significant correlation between absolute abundances of mRNAs and proteins has helped identify low abundance of proteins as the major limitation in peptide detection. Furthermore, we have discovered that iTRAQ labeling for quantitative proteomic analysis introduces a significant bias in peptide detection by mass spectrometry. Therefore, despite identifying at least one proteotypic peptide for almost all proteins in the PA, a context-dependent selection of proteotypic peptides appears to be the most effective approach for targeted proteomics.

iAB-RBC-283: A proteomically derived knowledge-base of erythrocyte metabolism that can be used to simulate its physiological and patho-physiological states.

Science.gov (United States)

Bordbar, Aarash; Jamshidi, Neema; Palsson, Bernhard O

2011-07-12

The development of high-throughput technologies capable of whole cell measurements of genes, proteins, and metabolites has led to the emergence of systems biology. Integrated analysis of the resulting omic data sets has proved to be hard to achieve. Metabolic network reconstructions enable complex relationships amongst molecular components to be represented formally in a biologically relevant manner while respecting physical constraints. In silico models derived from such reconstructions can then be queried or interrogated through mathematical simulations. Proteomic profiling studies of the mature human erythrocyte have shown more proteins present related to metabolic function than previously thought; however the significance and the causal consequences of these findings have not been explored. Erythrocyte proteomic data was used to reconstruct the most expansive description of erythrocyte metabolism to date, following extensive manual curation, assessment of the literature, and functional testing. The reconstruction contains 281 enzymes representing functions from glycolysis to cofactor and amino acid metabolism. Such a comprehensive view of erythrocyte metabolism implicates the erythrocyte as a potential biomarker for different diseases as well as a 'cell-based' drug-screening tool. The analysis shows that 94 erythrocyte enzymes are implicated in morbid single nucleotide polymorphisms, representing 142 pathologies. In addition, over 230 FDA-approved and experimental pharmaceuticals have enzymatic targets in the erythrocyte. The advancement of proteomic technologies and increased generation of high-throughput proteomic data have created the need for a means to analyze these data in a coherent manner. Network reconstructions provide a systematic means to integrate and analyze proteomic data in a biologically meaning manner. Analysis of the red cell proteome has revealed an unexpected level of complexity in the functional capabilities of human erythrocyte metabolism.

Plasma Proteome Biomarkers of Inflammation in School Aged Children in Nepal.

Directory of Open Access Journals (Sweden)

Sun Eun Lee

Full Text Available Inflammation is a condition stemming from complex host defense and tissue repair mechanisms, often simply characterized by plasma levels of a single acute reactant. We attempted to identify candidate biomarkers of systemic inflammation within the plasma proteome. We applied quantitative proteomics using isobaric mass tags (iTRAQ tandem mass spectrometry to quantify proteins in plasma of 500 Nepalese children 6-8 years of age. We evaluated those that co-vary with inflammation, indexed by α-1-acid glycoprotein (AGP, a conventional biomarker of inflammation in population studies. Among 982 proteins quantified in >10% of samples, 99 were strongly associated with AGP at a family-wise error rate of 0.1%. Magnitude and significance of association varied more among proteins positively (n = 41 than negatively associated (n = 58 with AGP. The former included known positive acute phase proteins including C-reactive protein, serum amyloid A, complement components, protease inhibitors, transport proteins with anti-oxidative activity, and numerous unexpected intracellular signaling molecules. Negatively associated proteins exhibited distinct differences in abundance between secretory hepatic proteins involved in transporting or binding lipids, micronutrients (vitamin A and calcium, growth factors and sex hormones, and proteins of largely extra-hepatic origin involved in the formation and metabolic regulation of extracellular matrix. With the same analytical approach and the significance threshold, seventy-two out of the 99 proteins were commonly associated with CRP, an established biomarker of inflammation, suggesting the validity of the identified proteins. Our findings have revealed a vast plasma proteome within a free-living population of children that comprise functional biomarkers of homeostatic and induced host defense, nutrient metabolism and tissue repair, representing a set of plasma proteins that may be used to assess dynamics and extent of

Extracellular membrane vesicles in blood products-biology and clinical relevance

Directory of Open Access Journals (Sweden)

Emilija Krstova Krajnc

2016-01-01

Full Text Available Extracellular membrane vesicles are fragments shed from plasma membranes off all cell types that are undergoing apoptosis or are being subjected to various types of stimulation or stress.  Even in the process of programmed cell death (apoptosis, cell fall apart of varying size vesicles. They expose phosphatidylserine (PS on the outer leaflet of their membrane, and bear surface membrane antigens reflecting their cellular origin. Extracellular membrane vesicles have been isolated from many types of biological fluids, including serum, cerebrospinal fluid, urine, saliva, tears and conditioned culture medium. Flow cytometry is one of the many different methodological approaches that have been used to analyze EMVs. The method attempts to characterize the EMVs cellular origin, size, population, number, and structure. EMVs are present and accumulate in blood products (erythrocytes, platelets as well as in fresh frozen plasma during storage. The aim of this review is to highlight the importance of extracellular vesicles as a cell-to-cell communication system and the role in the pathogenesis of different diseases. Special emphasis will be given to the implication of extracellular membrane vesicles in blood products and their clinical relevance. Although our understanding of the role of  EMVs in disease is far from comprehensive, they display promise as biomarkers for different diseases in the future and also as a marker of quality and safety in the quality control of blood products.

Network-based analysis of proteomic profiles

KAUST Repository

Wong, Limsoon

2016-01-26

Mass spectrometry (MS)-based proteomics is a widely used and powerful tool for profiling systems-wide protein expression changes. It can be applied for various purposes, e.g. biomarker discovery in diseases and study of drug responses. Although RNA-based high-throughput methods have been useful in providing glimpses into the underlying molecular processes, the evidences they provide are indirect. Furthermore, RNA and corresponding protein levels have been known to have poor correlation. On the other hand, MS-based proteomics tend to have consistency issues (poor reproducibility and inter-sample agreement) and coverage issues (inability to detect the entire proteome) that need to be urgently addressed. In this talk, I will discuss how these issues can be addressed by proteomic profile analysis techniques that use biological networks (especially protein complexes) as the biological context. In particular, I will describe several techniques that we have been developing for network-based analysis of proteomics profile. And I will present evidence that these techniques are useful in identifying proteomics-profile analysis results that are more consistent, more reproducible, and more biologically coherent, and that these techniques allow expansion of the detected proteome to uncover and/or discover novel proteins.

Arabidopsis peroxisome proteomics

Directory of Open Access Journals (Sweden)

John D. Bussell

2013-04-01

Full Text Available The analytical depth of investigation of the peroxisomal proteome of the model plant Arabidopsis thaliana has not yet reached that of other major cellular organelles such as chloroplasts or mitochondria. This is primarily due to the difficulties associated with isolating and obtaining purified samples of peroxisomes from Arabidopsis. So far only a handful of research groups have been successful in obtaining such fractions. To make things worse, enriched peroxisome fractions frequently suffer from significant organellar contamination, lowering confidence in localization assignment of the identified proteins. As with other cellular compartments, identification of peroxisomal proteins forms the basis for investigations of the dynamics of the peroxisomal proteome. It is therefore not surprising that, in terms of functional analyses by proteomic means, there remains a considerable gap between peroxisomes and chloroplasts or mitochondria. Alternative strategies are needed to overcome the obstacle of hard-to-obtain organellar fractions. This will help to close the knowledge gap between peroxisomes and other organelles and provide a full picture of the physiological pathways shared between organelles. In this review we briefly summarize the status quo and discuss some of the methodological alternatives to classic organelle proteomic approaches.

Differential Proteomic Analysis of Syncytiotrophoblast Extracellular Vesicles from Early-Onset Severe Preeclampsia, using 8-Plex iTRAQ Labeling Coupled with 2D Nano LC-MS/MS

Directory of Open Access Journals (Sweden)

Hongmei Li

2015-06-01

Full Text Available Aims: Previous studies have revealed that the increased shedding of syncytiotrophoblast extracellular vesicles (STBM may lead to preeclampsia (PE. We aimed to identify the proteins carried by STBM and their potential pathological roles in early-onset severe PE. Methods: In this study, we performed a differential proteomic analysis of STBM from early-onset severe PE patients, using iTRAQ isobaric tags and 2D nano LC-MS/MS. STBM were generated by the in vitro explant culture method, and then verified by electron microscopy and western blot analysis. Results: A total of 18 533 unique peptides and 3 317 proteins were identified, 3 292 proteins were quantified. We identified 194 differentially expressed proteins in STBM from early-onset severe PE patients, 122 proteins were up-regulated and 72 proteins were down-regulated. Further bioinformatics analysis revealed that mitochondrion, transmembrane transport and transmembrane transporter activity were the most abundant categories in gene ontology (GO annotation. Glycolysis/ gluconeogenesis, citrate cycle, fatty acid elongation, steroid hormone biosynthesis and oxidative phosphorylation were the five significantly represented pathways. Four differentially expressed proteins (siglec-6, calnexin, CD63 and S100-A8 related to inflammation, coagulation or immunoregulation were independently verified using western blot. Conclusions: The identification of key proteins carried by STBM may serve not only as a basis for better understanding and further exploring the etiology and pathogenesis of PE, but also as potential biomarkers and in providing targets for future therapy in PE, especially in early-onset severe PE(sPE.

Redox Proteomics and Platelet Activation: Understanding the Redox Proteome to Improve Platelet Quality for Transfusion

Science.gov (United States)

Sonego, Giona; Abonnenc, Mélanie; Tissot, Jean-Daniel; Prudent, Michel; Lion, Niels

2017-01-01

Blood banks use pathogen inactivation (PI) technologies to increase the safety of platelet concentrates (PCs). The characteristics of PI-treated PCs slightly differ from those of untreated PCs, but the underlying reasons are not well understood. One possible cause is the generation of oxidative stress during the PI process. This is of great interest since reactive oxygen species (ROS) act as second messengers in platelet functions. Furthermore, there are links between protein oxidation and phosphorylation, another mechanism that is critical for cell regulation. Current research efforts focus on understanding the underlying mechanisms and identifying new target proteins. Proteomics technologies represent powerful tools for investigating signaling pathways involving ROS and post-translational modifications such as phosphorylation, while quantitative techniques enable the comparison of the platelet resting state versus the stimulated state. In particular, redox cysteine is a key player in platelet activation upon stimulation by different agonists. This review highlights the experiments that have provided insights into the roles of ROS in platelet function and the implications for platelet transfusion, and potentially in diseases such as inflammation and platelet hyperactivity. The review also describes the implication of redox mechanism in platelet storage considerations. PMID:28208668

Extracellular matrix structure.

Science.gov (United States)

Theocharis, Achilleas D; Skandalis, Spyros S; Gialeli, Chrysostomi; Karamanos, Nikos K

2016-02-01

Extracellular matrix (ECM) is a non-cellular three-dimensional macromolecular network composed of collagens, proteoglycans/glycosaminoglycans, elastin, fibronectin, laminins, and several other glycoproteins. Matrix components bind each other as well as cell adhesion receptors forming a complex network into which cells reside in all tissues and organs. Cell surface receptors transduce signals into cells from ECM, which regulate diverse cellular functions, such as survival, growth, migration, and differentiation, and are vital for maintaining normal homeostasis. ECM is a highly dynamic structural network that continuously undergoes remodeling mediated by several matrix-degrading enzymes during normal and pathological conditions. Deregulation of ECM composition and structure is associated with the development and progression of several pathologic conditions. This article emphasizes in the complex ECM structure as to provide a better understanding of its dynamic structural and functional multipotency. Where relevant, the implication of the various families of ECM macromolecules in health and disease is also presented. Copyright © 2015 Elsevier B.V. All rights reserved.

Low Concentrations of Vitamin C Reduce the Synthesis of Extracellular Polymers and Destabilize Bacterial Biofilms

KAUST Repository

Pandit, Santosh

2017-12-26

Extracellular polymeric substances (EPS) produced by bacteria form a matrix supporting the complex three-dimensional architecture of biofilms. This EPS matrix is primarily composed of polysaccharides, proteins and extracellular DNA. In addition to supporting the community structure, the EPS matrix protects bacterial biofilms from the environment. Specifically, it shields the bacterial cells inside the biofilm, by preventing antimicrobial agents from getting in contact with them, thereby reducing their killing effect. New strategies for disrupting the formation of the EPS matrix can therefore lead to a more efficient use of existing antimicrobials. Here we examined the mechanism of the known effect of vitamin C (sodium ascorbate) on enhancing the activity of various antibacterial agents. Our quantitative proteomics analysis shows that non-lethal concentrations of vitamin C inhibit bacterial quorum sensing and other regulatory mechanisms underpinning biofilm development. As a result, the EPS biosynthesis in reduced, and especially the polysaccharide component of the matrix is depleted. Once the EPS content is reduced beyond a critical point, bacterial cells get fully exposed to the medium. At this stage, the cells are more susceptible to killing, either by vitamin C-induced oxidative stress as reported here, or by other antimicrobials or treatments.

Low Concentrations of Vitamin C Reduce the Synthesis of Extracellular Polymers and Destabilize Bacterial Biofilms

KAUST Repository

Pandit, Santosh; Ravikumar, Vaishnavi; Abdel-Haleem, Alyaa M.; Derouiche, Abderahmane; Mokkapati, V. R. S. S.; Sihlbom, Carina; Mineta, Katsuhiko; Gojobori, Takashi; Gao, Xin; Westerlund, Fredrik; Mijakovic, Ivan

2017-01-01

Extracellular polymeric substances (EPS) produced by bacteria form a matrix supporting the complex three-dimensional architecture of biofilms. This EPS matrix is primarily composed of polysaccharides, proteins and extracellular DNA. In addition to supporting the community structure, the EPS matrix protects bacterial biofilms from the environment. Specifically, it shields the bacterial cells inside the biofilm, by preventing antimicrobial agents from getting in contact with them, thereby reducing their killing effect. New strategies for disrupting the formation of the EPS matrix can therefore lead to a more efficient use of existing antimicrobials. Here we examined the mechanism of the known effect of vitamin C (sodium ascorbate) on enhancing the activity of various antibacterial agents. Our quantitative proteomics analysis shows that non-lethal concentrations of vitamin C inhibit bacterial quorum sensing and other regulatory mechanisms underpinning biofilm development. As a result, the EPS biosynthesis in reduced, and especially the polysaccharide component of the matrix is depleted. Once the EPS content is reduced beyond a critical point, bacterial cells get fully exposed to the medium. At this stage, the cells are more susceptible to killing, either by vitamin C-induced oxidative stress as reported here, or by other antimicrobials or treatments.

Acidosis-Induced Changes in Proteome Patterns of the Prostate Cancer-Derived Tumor Cell Line AT-1.

Science.gov (United States)

Ihling, Angelika; Ihling, Christian H; Sinz, Andrea; Gekle, Michael

2015-09-04

Under various pathological conditions, such as inflammation, ischemia and in solid tumors, physiological parameters (local oxygen tension or extracellular pH) show distinct tissue abnormalities (hypoxia and acidosis). For tumors, the prevailing microenvironment exerts a strong influence on the phenotype with respect to proliferation, invasion, and metastasis formation and therefore influences prognosis. In this study, we investigate the impact of extracellular metabolic acidosis (pH 7.4 versus 6.6) on the proteome patterns of a prostate cancer-derived tumor cell type (AT-1) using isobaric labeling and LC-MS/MS analysis. In total, 2710 proteins were identified and quantified across four biological replicates, of which seven were significantly affected with changes >50% and used for validation. Glucose transporter 1 and farnesyl pyrophosphatase were found to be down-regulated after 48 h of acidic treatment, and metallothionein 2A was reduced after 24 h and returned to control values after 48 h. After 24 and 48 h at pH 6.6, glutathione S transferase A3 and NAD(P)H dehydrogenase 1, cellular retinoic acid-binding protein 2, and Na-bicarbonate transporter 3 levels were found to be increased. The changes in protein levels were confirmed by transcriptome and functional analyses. In addition to the experimental in-depth investigation of proteins with changes >50%, functional profiling (statistical enrichment analysis) including proteins with changes >20% revealed that acidosis upregulates GSH metabolic processes, citric acid cycle, and respiratory electron transport. Metabolism of lipids and cholesterol biosynthesis were downregulated. Our data comprise the first comprehensive report on acidosis-induced changes in proteome patterns of a tumor cell line.

Proteomics Insights into Autophagy.

Science.gov (United States)

Cudjoe, Emmanuel K; Saleh, Tareq; Hawkridge, Adam M; Gewirtz, David A

2017-10-01

Autophagy, a conserved cellular process by which cells recycle their contents either to maintain basal homeostasis or in response to external stimuli, has for the past two decades become one of the most studied physiological processes in cell biology. The 2016 Nobel Prize in Medicine and Biology awarded to Dr. Ohsumi Yoshinori, one of the first scientists to characterize this cellular mechanism, attests to its importance. The induction and consequent completion of the process of autophagy results in wide ranging changes to the cellular proteome as well as the secretome. MS-based proteomics affords the ability to measure, in an unbiased manner, the ubiquitous changes that occur when autophagy is initiated and progresses in the cell. The continuous improvements and advances in mass spectrometers, especially relating to ionization sources and detectors, coupled with advances in proteomics experimental design, has made it possible to study autophagy, among other process, in great detail. Innovative labeling strategies and protein separation techniques as well as complementary methods including immuno-capture/blotting/staining have been used in proteomics studies to provide more specific protein identification. In this review, we will discuss recent advances in proteomics studies focused on autophagy. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitative proteomics of fractionated membrane and lumen exosome proteins from isogenic metastatic and nonmetastatic bladder cancer cells reveal differential expression of EMT factors

DEFF Research Database (Denmark)

Jeppesen, Dennis Kjølhede; Nawrocki, Arkadiusz; Jensen, Steffen Grann

2014-01-01

Cancer cells secrete soluble factors and various extracellular vesicles, including exosomes, into their tissue microenvironment. The secretion of exosomes is speculated to facilitate local invasion and metastatic spread. Here, we used an in vivo metastasis model of human bladder carcinoma cell line...... T24 without metastatic capacity and its two isogenic derivate cell lines SLT4 and FL3, which form metastases in the lungs and liver of mice, respectively. Cultivation in CLAD1000 bioreactors rather than conventional culture flasks resulted in a 13-16-fold increased exosome yield and facilitated...... quantitative proteomics of fractionated exosomes. Exosomes from T24, SLT4, and FL3 cells were partitioned into membrane and luminal fractions and changes in protein abundance related to the gain of metastatic capacity were identified by quantitative iTRAQ- proteomics. We identified several proteins linked...

In-depth proteomics characterization of embryogenesis of the honey bee worker (Apis mellifera ligustica).

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Fang, Yu; Feng, Mao; Han, Bin; Lu, Xiaoshan; Ramadan, Haitham; Li, Jianke

2014-09-01

Identifying proteome changes of honey bee embryogenesis is of prime importance for unraveling the molecular mechanisms that they underlie. However, many proteomic changes during the embryonic period are not well characterized. We analyzed the proteomic alterations over the complete time course of honey bee worker embryogenesis at 24, 48, and 72 h of age, using mass spectrometry-based proteomics, label-free quantitation, and bioinformatics. Of the 1460 proteins identified the embryo of all three ages, the core proteome (proteins shared by the embryos of all three ages, accounting for 40%) was mainly involved in protein synthesis, metabolic energy, development, and molecular transporter, which indicates their centrality in driving embryogenesis. However, embryos at different developmental stages have their own specific proteome and pathway signatures to coordinate and modulate developmental events. The young embryos (proteins related to nutrition storage and nucleic acid metabolism may correlate with the cell proliferation occurring at this stage. The middle aged embryos (24-48 h) enhanced expression of proteins associated with cell cycle control, transporters, antioxidant activity, and the cytoskeleton suggest their roles to support rudimentary organogenesis. Among these proteins, the biological pathways of aminoacyl-tRNA biosynthesis, β-alanine metabolism, and protein export are intensively activated in the embryos of middle age. The old embryos (48-72 h) elevated expression of proteins implicated in fatty acid metabolism and morphogenesis indicate their functionality for the formation and development of organs and dorsal closure, in which the biological pathways of fatty acid metabolism and RNA transport are highly activated. These findings add novel understanding to the molecular details of honey bee embryogenesis, in which the programmed activation of the proteome matches with the physiological transition observed during embryogenesis. The identified biological

Extracellular small heat shock proteins: exosomal biogenesis and function.

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Reddy, V Sudhakar; Madala, Satish K; Trinath, Jamma; Reddy, G Bhanuprakash

2018-05-01

Small heat shock proteins (sHsps) belong to the family of heat shock proteins (Hsps): some are induced in response to multiple stressful events to protect the cells while others are constitutively expressed. Until now, it was believed that Hsps, including sHsps, are present inside the cells and perform intracellular functions. Interestingly, several groups recently reported the extracellular presence of Hsps, and sHsps have also been detected in sera/cerebrospinal fluids in various pathological conditions. Secretion into the extracellular milieu during many pathological conditions suggests additional or novel functions of sHsps in addition to their intracellular properties. Extracellular sHsps are implicated in cell-cell communication, activation of immune cells, and promoting anti-inflammatory and anti-platelet responses. Interestingly, exogenous administration of sHsps showed therapeutic effects in multiple disease models implying that extracellular sHsps are beneficial in pathological conditions. sHsps do not possess signal sequence and, hence, are not exported through the classical Endoplasmic reticulum-Golgi complex (ER-Golgi) secretory pathway. Further, export of sHsps is not inhibited by ER-Golgi secretory pathway inhibitors implying the involvement of a nonclassical secretory pathway in sHsp export. In lieu, lysoendosomal and exosomal pathways have been proposed for the export of sHsps. Heat shock protein 27 (Hsp27), αB-crystallin (αBC), and Hsp20 are shown to be exported by exosomes. Exosomes packaged with sHsps have beneficial effects in in vivo disease models. However, secretion mechanisms and therapeutic use of sHsps have not been elucidated in detail. Therefore, this review aimed at highlighting the current understanding of sHsps (Hsp27, αBC, and Hsp20) in the extracellular medium.

Identification of pathogenic factors potentially involved in Staphylococcus aureus keratitis using proteomics.

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Khan, Shamila; Cole, Nerida; Hume, Emma B H; Garthwaite, Linda L; Nguyen-Khuong, Terry; Walsh, Bradley J; Willcox, Mark D P

2016-10-01

Staphylococcus is a leading cause of microbial keratitis, characterized by destruction of the cornea by bacterial exoproteins and host-associated factors. The aim of this study was to compare extracellular and cell-associated proteins produced by two different isolates of S. aureus, a virulent clinical isolate (Staph 38) and a laboratory strain (Staphylococcus aureus 8325-4) of weaker virulence in the mouse keratitis model. Proteins were analyzed using 2D polyacrylamide gel electrophoresis and identified by subsequent mass spectrometry. Activity of staphylococcal adhesins was assessed by allowing strains to bind to various proteins adsorbed onto polymethylmethacrylate squares. Thirteen proteins in the extracellular fraction and eight proteins in the cell-associated fractions after bacterial growth were produced in increased amounts in the clinical isolate Staph 38. Four of these proteins were S. aureus virulence factor adhesins, fibronectin binding protein A, staphopain, glyceraldehyde-3-phosphate dehydrogenase 2 and extracellular adherence protein. The clinical isolate Staph 38 adhered to a greater extent to all mammalian proteins tested, indicating the potential of the adhesins to be active on its surface. Other proteins with increased expression in Staph 38 included potential moonlighting proteins and proteins involved in transcription or translation. This is the first demonstration of the proteome of S. aureus isolates from keratitis. These results indicate that the virulent clinical isolate produces more potentially important virulence factors compared to the less virulent laboratory strain and these may be associated with the ability of a S. aureus strain to cause more severe keratitis. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Progress in stable isotope labeled quantitative proteomics methods].

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Zhou, Yuan; Shan, Yichu; Zhang, Lihua; Zhang, Yukui

2013-06-01

Quantitative proteomics is an important research field in post-genomics era. There are two strategies for proteome quantification: label-free methods and stable isotope labeling methods which have become the most important strategy for quantitative proteomics at present. In the past few years, a number of quantitative methods have been developed, which support the fast development in biology research. In this work, we discuss the progress in the stable isotope labeling methods for quantitative proteomics including relative and absolute quantitative proteomics, and then give our opinions on the outlook of proteome quantification methods.

Proteomic analysis of the organic matrix of the abalone Haliotis asinina calcified shell

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Degnan Bernard M

2010-11-01

Full Text Available Abstract Background The formation of the molluscan shell is regulated to a large extent by a matrix of extracellular macromolecules that are secreted by the shell forming tissue, the mantle. This so called "calcifying matrix" is a complex mixture of proteins and glycoproteins that is assembled and occluded within the mineral phase during the calcification process. While the importance of the calcifying matrix to shell formation has long been appreciated, most of its protein components remain uncharacterised. Results Recent expressed sequence tag (EST investigations of the mantle tissue from the tropical abalone (Haliotis asinina provide an opportunity to further characterise the proteins in the shell by a proteomic approach. In this study, we have identified a total of 14 proteins from distinct calcified layers of the shell. Only two of these proteins have been previously characterised from abalone shells. Among the novel proteins are several glutamine- and methionine-rich motifs and hydrophobic glycine-, alanine- and acidic aspartate-rich domains. In addition, two of the new proteins contained Kunitz-like and WAP (whey acidic protein protease inhibitor domains. Conclusion This is one of the first comprehensive proteomic study of a molluscan shell, and should provide a platform for further characterization of matrix protein functions and interactions.

NIH Common Fund - Disruptive Proteomics Technologies - Challenges and Opportunities | Office of Cancer Clinical Proteomics Research

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This Request for Information (RFI) is directed toward determining how best to accelerate research in disruptive proteomics technologies. The Disruptive Proteomics Technologies (DPT) Working Group of the NIH Common Fund wishes to identify gaps and opportunities in current technologies and methodologies related to proteome-wide measurements.  For the purposes of this RFI, “disruptive” is defined as very rapid, very significant gains, similar to the "disruptive" technology development that occurred in DNA sequencing technology.

Quantitative proteomics of Spodoptera frugiperda cells during growth and baculovirus infection.

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Nuno Carinhas

Full Text Available Baculovirus infection of Spodoptera frugiperda cells is a system of choice to produce a range of recombinant proteins, vaccines and, potentially, gene therapy vectors. While baculovirus genomes are well characterized, the genome of S. frugiperda is not sequenced and the virus-host molecular interplay is sparsely known. Herein, we describe the application of stable isotope labeling by amino acids in cell culture (SILAC to obtain the first comparative proteome quantitation of S. frugiperda cells during growth and early baculovirus infection. The proteome coverage was maximized by compiling a search database with protein annotations from insect species. Of interest were differentially proteins related to energy metabolism, endoplasmic reticulum and oxidative stress, yet not investigated in the scope of baculovirus infection. Further, the reduced expression of key viral-encoded proteins early in the infection cycle is suggested to be related with decreased viral replication at high cell density culture. These findings have implications for virological research and improvement of baculovirus-based bioprocesses.

A decade of proteomics accomplished! Central and Eastern European Proteomic Conference (CEEPC) celebrates its 10th Anniversary in Budapest, Hungary.

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Gadher, Suresh Jivan; Drahos, László; Vékey, Károly; Kovarova, Hana

2017-07-01

The Central and Eastern European Proteomic Conference (CEEPC) proudly celebrated its 10th Anniversary with an exciting scientific program inclusive of proteome, proteomics and systems biology in Budapest, Hungary. Since 2007, CEEPC has represented 'state-of the-art' proteomics in and around Central and Eastern Europe and these series of conferences have become a well-recognized event in the proteomic calendar. Fresher challenges and global healthcare issues such as ageing and chronic diseases are driving clinical and scientific research towards regenerative, reparative and personalized medicine. To this end, proteomics may enable diverse intertwining research fields to reach their end goals. CEEPC will endeavor to facilitate these goals.

Extracellular Vesicle-Associated Transitory Cell Wall Components and Their Impact on the Interaction of Fungi with Host Cells.

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Nimrichter, Leonardo; de Souza, Marcio M; Del Poeta, Maurizio; Nosanchuk, Joshua D; Joffe, Luna; Tavares, Patricia de M; Rodrigues, Marcio L

2016-01-01

Classic cell wall components of fungi comprise the polysaccharides glucans and chitin, in association with glycoproteins and pigments. During the last decade, however, system biology approaches clearly demonstrated that the composition of fungal cell walls include atypical molecules historically associated with intracellular or membrane locations. Elucidation of mechanisms by which many fungal molecules are exported to the extracellular space suggested that these atypical components are transitorily located to the cell wall. The presence of extracellular vesicles (EVs) at the fungal cell wall and in culture supernatants of distinct pathogenic species suggested a highly functional mechanism of molecular export in these organisms. Thus, the passage of EVs through fungal cell walls suggests remarkable molecular diversity and, consequently, a potentially variable influence on the host antifungal response. On the basis of information derived from the proteomic characterization of fungal EVs from the yeasts Cryptoccocus neoformans and Candida albicans and the dimorphic fungi Histoplasma capsulatum and Paracoccidioides brasiliensis, our manuscript is focused on the clear view that the fungal cell wall is much more complex than previously thought.

Proteomics in pulmonary research: selected methodical aspects

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Martin Petrek

2007-10-01

Full Text Available Recent years witness rapid expansion of applications of proteomics to clinical research including non-malignant lung disorders. These developments bring along the need for standardisation of proteomic experiments. This paper briefly reviews basic methodical aspects of appliedproteomic studies using SELDI-TOF mass spectrometry platform as example but also emphasizes general aspects of quality assurance in proteomics. Key-words: lung proteome, quality assurance, SELDI-TOF MS

The potato tuber mitochondrial proteome

DEFF Research Database (Denmark)

Salvato, Fernanda; Havelund, Jesper Foged; Chen, Mingjie

2014-01-01

Mitochondria are called the powerhouses of the cell. To better understand the role of mitochondria in maintaining and regulating metabolism in storage tissues, highly purified mitochondria were isolated from dormant potato tubers (Solanum tuberosum 'Folva') and their proteome investigated. Proteins...... manner using normalized spectral counts including as many as 5-fold more "extreme" proteins (low mass, high isoelectric point, hydrophobic) than previous mitochondrial proteome studies. We estimate that this compendium of proteins represents a high coverage of the potato tuber mitochondrial proteome...

iAB-RBC-283: A proteomically derived knowledge-base of erythrocyte metabolism that can be used to simulate its physiological and patho-physiological states

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Palsson Bernhard O

2011-07-01

Full Text Available Abstract Background The development of high-throughput technologies capable of whole cell measurements of genes, proteins, and metabolites has led to the emergence of systems biology. Integrated analysis of the resulting omic data sets has proved to be hard to achieve. Metabolic network reconstructions enable complex relationships amongst molecular components to be represented formally in a biologically relevant manner while respecting physical constraints. In silico models derived from such reconstructions can then be queried or interrogated through mathematical simulations. Proteomic profiling studies of the mature human erythrocyte have shown more proteins present related to metabolic function than previously thought; however the significance and the causal consequences of these findings have not been explored. Results Erythrocyte proteomic data was used to reconstruct the most expansive description of erythrocyte metabolism to date, following extensive manual curation, assessment of the literature, and functional testing. The reconstruction contains 281 enzymes representing functions from glycolysis to cofactor and amino acid metabolism. Such a comprehensive view of erythrocyte metabolism implicates the erythrocyte as a potential biomarker for different diseases as well as a 'cell-based' drug-screening tool. The analysis shows that 94 erythrocyte enzymes are implicated in morbid single nucleotide polymorphisms, representing 142 pathologies. In addition, over 230 FDA-approved and experimental pharmaceuticals have enzymatic targets in the erythrocyte. Conclusion The advancement of proteomic technologies and increased generation of high-throughput proteomic data have created the need for a means to analyze these data in a coherent manner. Network reconstructions provide a systematic means to integrate and analyze proteomic data in a biologically meaning manner. Analysis of the red cell proteome has revealed an unexpected level of complexity in

Proteome profile of swine testicular cells infected with porcine transmissible gastroenteritis coronavirus.

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Ruili Ma

Full Text Available The interactions occurring between a virus and a host cell during a viral infection are complex. The purpose of this paper was to analyze altered cellular protein levels in porcine transmissible gastroenteritis coronavirus (TGEV-infected swine testicular (ST cells in order to determine potential virus-host interactions. A proteomic approach using isobaric tags for relative and absolute quantitation (iTRAQ-coupled two-dimensional liquid chromatography-tandem mass spectrometry identification was conducted on the TGEV-infected ST cells. The results showed that the 4-plex iTRAQ-based quantitative approach identified 4,112 proteins, 146 of which showed significant changes in expression 48 h after infection. At 64 h post infection, 219 of these proteins showed significant change, further indicating that a larger number of proteomic changes appear to occur during the later stages of infection. Gene ontology analysis of the altered proteins showed enrichment in multiple biological processes, including cell adhesion, response to stress, generation of precursor metabolites and energy, cell motility, protein complex assembly, growth, developmental maturation, immune system process, extracellular matrix organization, locomotion, cell-cell signaling, neurological system process, and cell junction organization. Changes in the expression levels of transforming growth factor beta 1 (TGF-β1, caspase-8, and heat shock protein 90 alpha (HSP90α were also verified by western blot analysis. To our knowledge, this study is the first time the response profile of ST host cells following TGEV infection has been analyzed using iTRAQ technology, and our description of the late proteomic changes that are occurring after the time of vigorous viral production are novel. Therefore, this study provides a solid foundation for further investigation, and will likely help us to better understand the mechanisms of TGEV infection and pathogenesis.

Red blood cell (RBC) membrane proteomics--Part I: Proteomics and RBC physiology.

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Pasini, Erica M; Lutz, Hans U; Mann, Matthias; Thomas, Alan W

2010-01-03

Membrane proteomics is concerned with accurately and sensitively identifying molecules involved in cell compartmentalisation, including those controlling the interface between the cell and the outside world. The high lipid content of the environment in which these proteins are found often causes a particular set of problems that must be overcome when isolating the required material before effective HPLC-MS approaches can be performed. The membrane is an unusually dynamic cellular structure since it interacts with an ever changing environment. A full understanding of this critical cell component will ultimately require, in addition to proteomics, lipidomics, glycomics, interactomics and study of post-translational modifications. Devoid of nucleus and organelles in mammalian species other than camelids, and constantly in motion in the blood stream, red blood cells (RBCs) are the sole mammalian oxygen transporter. The fact that mature mammalian RBCs have no internal membrane-bound organelles, somewhat simplifies proteomics analysis of the plasma membrane and the fact that it has no nucleus disqualifies microarray based methods. Proteomics has the potential to provide a better understanding of this critical interface, and thereby assist in identifying new approaches to diseases. (c) 2009 Elsevier B.V. All rights reserved.

Proteomic Analysis of Chinese Hamster Ovary Cells

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Baycin-Hizal, Deniz; Tabb, David L.; Chaerkady, Raghothama

2012-01-01

To complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO cells including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis, multidimens......To complement the recent genomic sequencing of Chinese hamster ovary (CHO) cells, proteomic analysis was performed on CHO cells including the cellular proteome, secretome, and glycoproteome using tandem mass spectrometry (MS/MS) of multiple fractions obtained from gel electrophoresis...

Semen proteomics and male infertility.

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Jodar, Meritxell; Soler-Ventura, Ada; Oliva, Rafael

2017-06-06

Semen is a complex body fluid containing an admixture of spermatozoa suspended in secretions from the testes and epididymis which are mixed at the time of ejaculation with secretions from other accessory sex glands such as the prostate and seminal vesicles. High-throughput technologies have revealed that, contrary to the idea that sperm cells are simply a silent delivery vehicle of the male genome to the oocyte, the sperm cells in fact provide both a specific epigenetically marked DNA together with a complex population of proteins and RNAs crucial for embryogenesis. Similarly, -omic technologies have also enlightened that seminal fluid seems to play a much greater role than simply being a medium to carry the spermatozoa through the female reproductive tract. In the present review, we briefly overview the sperm cell biology, consider the key issues in sperm and seminal fluid sample preparation for high-throughput proteomic studies, describe the current state of the sperm and seminal fluid proteomes generated by high-throughput proteomic technologies and provide new insights into the potential communication between sperm and seminal fluid. In addition, comparative proteomic studies open a window to explore the potential pathogenic mechanisms of infertility and the discovery of potential biomarkers with clinical significance. The review updates the numerous proteomics studies performed on semen, including spermatozoa and seminal fluid. In addition, an integrative analysis of the testes, sperm and seminal fluid proteomes is also included providing insights into the molecular mechanisms that regulate the generation, maturation and transit of spermatozoa. Furthermore, the compilation of several differential proteomic studies focused on male infertility reveals potential pathways disturbed in specific subtypes of male infertility and points out towards future research directions in the field. Copyright © 2016 Elsevier B.V. All rights reserved.

Progress on the HUPO Draft Human Proteome: 2017 Metrics of the Human Proteome Project.

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Omenn, Gilbert S; Lane, Lydie; Lundberg, Emma K; Overall, Christopher M; Deutsch, Eric W

2017-12-01

The Human Proteome Organization (HUPO) Human Proteome Project (HPP) continues to make progress on its two overall goals: (1) completing the protein parts list, with an annual update of the HUPO draft human proteome, and (2) making proteomics an integrated complement to genomics and transcriptomics throughout biomedical and life sciences research. neXtProt version 2017-01-23 has 17 008 confident protein identifications (Protein Existence [PE] level 1) that are compliant with the HPP Guidelines v2.1 ( https://hupo.org/Guidelines ), up from 13 664 in 2012-12 and 16 518 in 2016-04. Remaining to be found by mass spectrometry and other methods are 2579 "missing proteins" (PE2+3+4), down from 2949 in 2016. PeptideAtlas 2017-01 has 15 173 canonical proteins, accounting for nearly all of the 15 290 PE1 proteins based on MS data. These resources have extensive data on PTMs, single amino acid variants, and splice isoforms. The Human Protein Atlas v16 has 10 492 highly curated protein entries with tissue and subcellular spatial localization of proteins and transcript expression. Organ-specific popular protein lists have been generated for broad use in quantitative targeted proteomics using SRM-MS or DIA-SWATH-MS studies of biology and disease.

Immunoregulation of Neutrophil Extracellular Trap Formation by Endothelial-Derived p33 (gC1q Receptor)

DEFF Research Database (Denmark)

Neumann, Ariane; Papareddy, Praveen; Westman, Johannes

2018-01-01

The formation of neutrophil extracellular traps (NETs) is a host defence mechanism, known to facilitate the entrapment and growth inhibition of many bacterial pathogens. It has been implicated that the translocation of myeloperoxidase (MPO) from neutrophilic granules to the nucleus is crucial to ...

Fibroblasts and the extracellular matrix in right ventricular disease.

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Frangogiannis, Nikolaos G

2017-10-01

Right ventricular failure predicts adverse outcome in patients with pulmonary hypertension (PH), and in subjects with left ventricular heart failure and is associated with interstitial fibrosis. This review manuscript discusses the cellular effectors and molecular mechanisms implicated in right ventricular fibrosis. The right ventricular interstitium contains vascular cells, fibroblasts, and immune cells, enmeshed in a collagen-based matrix. Right ventricular pressure overload in PH is associated with the expansion of the fibroblast population, myofibroblast activation, and secretion of extracellular matrix proteins. Mechanosensitive transduction of adrenergic signalling and stimulation of the renin-angiotensin-aldosterone cascade trigger the activation of right ventricular fibroblasts. Inflammatory cytokines and chemokines may contribute to expansion and activation of macrophages that may serve as a source of fibrogenic growth factors, such as transforming growth factor (TGF)-β. Endothelin-1, TGF-βs, and matricellular proteins co-operate to activate cardiac myofibroblasts, and promote synthesis of matrix proteins. In comparison with the left ventricle, the RV tolerates well volume overload and ischemia; whether the right ventricular interstitial cells and matrix are implicated in these favourable responses remains unknown. Expansion of fibroblasts and extracellular matrix protein deposition are prominent features of arrhythmogenic right ventricular cardiomyopathies and may be implicated in the pathogenesis of arrhythmic events. Prevailing conceptual paradigms on right ventricular remodelling are based on extrapolation of findings in models of left ventricular injury. Considering the unique embryologic, morphological, and physiologic properties of the RV and the clinical significance of right ventricular failure, there is a need further to dissect RV-specific mechanisms of fibrosis and interstitial remodelling. Published on behalf of the European Society of

Analysis of cellular and extracellular DNA in fingerprints

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Button, Julie M. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

2014-09-09

It has been previously shown that DNA can be recovered from latent fingerprints left on various surfaces [R. A. H. van Oorschot and M. K. Jones, Nature 387, 767 (1997)]. However, the source of the DNA, extracellular versus cellular origin, is difficult to determine. If the DNA is cellular, it is believed to belong to skin cells while extracellular DNA is believed to originate from body fluids such as sweat [D. J. Daly et. al, Forensic Sci. Int. Genet. 6, 41-46 (2012); V. V. Vlassov et. al, BioEssays 29, 654-667 (2007)]. The origin of the DNA in fingerprints has implications for processing and interpretation of forensic evidence. The determination of the origin of DNA in fingerprints is further complicated by the fact that the DNA in fingerprints tends to be at a very low quantity [R. A. H. van Oorschot and M. K. Jones, Nature 387, 767 (1997)]. This study examined fingerprints from five volunteers left on sterilized glass slides and plastic pens. Three fingerprints were left on each glass slide (thumb, index, and middle fingers) while the pens were held as if one was writing with them. The DNA was collected from the objects using the wet swabbing technique (TE buffer). Following collection, the cellular and extracellular components of each sample were separated using centrifugation and an acoustofluidics system. Centrifugation is still the primary separation technique utilized in forensics laboratories, while acoustic focusing uses sound waves to focus large particles (cells) into low pressure nodes, separating them from the rest of the sample matrix. After separation, all samples were quantified using real-time quantitative PCR (qPCR). The overall trend is that there is more DNA in the extracellular fractions than cellular fractions for both centrifugation and acoustofluidic processing. Additionally, more DNA was generally collected from the pen samples than the samples left on glass slides.

Comparative Tissue Proteomics of Microdissected Specimens Reveals Novel Candidate Biomarkers of Bladder Cancer*

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Chen, Chien-Lun; Chung, Ting; Wu, Chih-Ching; Ng, Kwai-Fong; Yu, Jau-Song; Tsai, Cheng-Han; Chang, Yu-Sun; Liang, Ying; Tsui, Ke-Hung; Chen, Yi-Ting

2015-01-01

More than 380,000 new cases of bladder cancer are diagnosed worldwide, accounting for ∼150,200 deaths each year. To discover potential biomarkers of bladder cancer, we employed a strategy combining laser microdissection, isobaric tags for relative and absolute quantitation labeling, and liquid chromatography-tandem MS (LC-MS/MS) analysis to profile proteomic changes in fresh-frozen bladder tumor specimens. Cellular proteins from four pairs of surgically resected primary bladder cancer tumor and adjacent nontumorous tissue were extracted for use in two batches of isobaric tags for relative and absolute quantitation experiments, which identified a total of 3220 proteins. A DAVID (database for annotation, visualization and integrated discovery) analysis of dysregulated proteins revealed that the three top-ranking biological processes were extracellular matrix organization, extracellular structure organization, and oxidation-reduction. Biological processes including response to organic substances, response to metal ions, and response to inorganic substances were highlighted by up-expressed proteins in bladder cancer. Seven differentially expressed proteins were selected as potential bladder cancer biomarkers for further verification. Immunohistochemical analyses showed significantly elevated levels of three proteins—SLC3A2, STMN1, and TAGLN2—in tumor cells compared with noncancerous bladder epithelial cells, and suggested that TAGLN2 could be a useful tumor tissue marker for diagnosis (AUC = 0.999) and evaluating lymph node metastasis in bladder cancer patients. ELISA results revealed significantly increased urinary levels of both STMN1 and TAGLN2 in bladder cancer subgroups compared with control groups. In comparisons with age-matched hernia urine specimens, urinary TAGLN2 in bladder cancer samples showed the largest fold change (7.13-fold), with an area-under-the-curve value of 0.70 (p < 0.001, n = 205). Overall, TAGLN2 showed the most significant

Data in support of the identification of neuronal and astrocyte proteins interacting with extracellularly applied oligomeric and fibrillar α-synuclein assemblies by mass spectrometry.

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Shrivastava, Amulya Nidhi; Redeker, Virginie; Fritz, Nicolas; Pieri, Laura; Almeida, Leandro G; Spolidoro, Maria; Liebmann, Thomas; Bousset, Luc; Renner, Marianne; Léna, Clément; Aperia, Anita; Melki, Ronald; Triller, Antoine

2016-06-01

α-Synuclein (α-syn) is the principal component of Lewy bodies, the pathophysiological hallmark of individuals affected by Parkinson disease (PD). This neuropathologic form of α-syn contributes to PD progression and propagation of α-syn assemblies between neurons. The data we present here support the proteomic analysis used to identify neuronal proteins that specifically interact with extracellularly applied oligomeric or fibrillar α-syn assemblies (conditions 1 and 2, respectively) (doi: 10.15252/embj.201591397[1]). α-syn assemblies and their cellular partner proteins were pulled down from neuronal cell lysed shortly after exposure to exogenous α-syn assemblies and the associated proteins were identified by mass spectrometry using a shotgun proteomic-based approach. We also performed experiments on pure cultures of astrocytes to identify astrocyte-specific proteins interacting with oligomeric or fibrillar α-syn (conditions 3 and 4, respectively). For each condition, proteins interacting selectively with α-syn assemblies were identified by comparison to proteins pulled-down from untreated cells used as controls. The mass spectrometry data, the database search and the peak lists have been deposited to the ProteomeXchange Consortium database via the PRIDE partner repository with the dataset identifiers PRIDE: PXD002256 to PRIDE: PXD002263 and doi: 10.6019/PXD002256 to 10.6019/PXD002263.

Light Regimes Shape Utilization of Extracellular Organic C and N in a Cyanobacterial Biofilm

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Stuart, Rhona K.; Mayali, Xavier; Boaro, Amy A.; Zemla, Adam; Everroad, R. Craig; Nilson, Daniel; Weber, Peter K.; Lipton, Mary; Bebout, Brad M.; Pett-Ridge, Jennifer; Thelen, Michael P.

2016-06-28

>IMPORTANCECyanobacteria are globally distributed primary producers, and the fate of their fixed C influences microbial biogeochemical cycling. This fate is complicated by cyanobacterial degradation and assimilation of organic matter, but because cyanobacteria are assumed to be poor competitors for organic matter consumption, regulation of this process is not well tested. In mats and biofilms, this is especially relevant because cyanobacteria produce an extensive organic extracellular matrix, providing the community with a rich source of nutrients. Light is a well-known regulator of cyanobacterial metabolism, so we characterized the effects of light availability on the incorporation of organic matter. Using stable isotope tracing at the single-cell level, we quantified photoautotroph assimilation under different metabolic conditions and integrated the results with proteomics to elucidate metabolic status. We found that cyanobacteria effectively compete for organic matter in the light and the dark and that nutrient requirements and community interactions contribute to cycling of extracellular organic matter.

Proteomic approaches in brain research and neuropharmacology.

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Vercauteren, Freya G G; Bergeron, John J M; Vandesande, Frans; Arckens, Lut; Quirion, Rémi

2004-10-01

Numerous applications of genomic technologies have enabled the assembly of unprecedented inventories of genes, expressed in cells under specific physiological and pathophysiological conditions. Complementing the valuable information generated through functional genomics with the integrative knowledge of protein expression and function should enable the development of more efficient diagnostic tools and therapeutic agents. Proteomic analyses are particularly suitable to elucidate posttranslational modifications, expression levels and protein-protein interactions of thousands of proteins at a time. In this review, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) investigations of brain tissues in neurodegenerative diseases such as Alzheimer's disease, Down syndrome and schizophrenia, and the construction of 2D-PAGE proteome maps of the brain are discussed. The role of the Human Proteome Organization (HUPO) as an international coordinating organization for proteomic efforts, as well as challenges for proteomic technologies and data analysis are also addressed. It is expected that the use of proteomic strategies will have significant impact in neuropharmacology over the coming decade.

Proteomic landscape in Central and Eastern Europe: the 9th Central and Eastern European Proteomic Conference, Poznan, Poland

Czech Academy of Sciences Publication Activity Database

Gadher, S. J.; Marczak, L.; Luczak, M.; Stobiecki, M.; Widlak, P.; Kovářová, Hana

2016-01-01

Roč. 13, č. 1 (2016), s. 5-7 ISSN 1478-9450. [Central and Eastern European Proteomic Conference (CEEPC) /9./. Poznaň, 15.06.2015-18.06.2015] Institutional support: RVO:67985904 Keywords : Central and Eastern Proteomic Conference * proteomics * mass spectrometry imaging Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.849, year: 2016

Protein Dynamics in the Plant Extracellular Space

Directory of Open Access Journals (Sweden)

Leonor Guerra-Guimarães

2016-07-01

Full Text Available The extracellular space (ECS or apoplast is the plant cell compartment external to the plasma membrane, which includes the cell walls, the intercellular space and the apoplastic fluid (APF. The present review is focused on APF proteomics papers and intends to draw information on the metabolic processes occurring in the ECS under abiotic and biotic stresses, as well as under non-challenged conditions. The large majority of the proteins detected are involved in “cell wall organization and biogenesis”, “response to stimulus” and “protein metabolism”. It becomes apparent that some proteins are always detected, irrespective of the experimental conditions, although with different relative contribution. This fact suggests that non-challenged plants have intrinsic constitutive metabolic processes of stress/defense in the ECS. In addition to the multiple functions ascribed to the ECS proteins, should be considered the interactions established between themselves and with the plasma membrane and its components. These interactions are crucial in connecting exterior and interior of the cell, and even simple protein actions in the ECS can have profound effects on plant performance. The proteins of the ECS are permanently contributing to the high dynamic nature of this plant compartment, which seems fundamental to plant development and adaptation to the environmental conditions.

Proteome-metabolome profiling of ovarian cancer ascites reveals novel components involved in intercellular communication.

Science.gov (United States)

Shender, Victoria O; Pavlyukov, Marat S; Ziganshin, Rustam H; Arapidi, Georgij P; Kovalchuk, Sergey I; Anikanov, Nikolay A; Altukhov, Ilya A; Alexeev, Dmitry G; Butenko, Ivan O; Shavarda, Alexey L; Khomyakova, Elena B; Evtushenko, Evgeniy; Ashrafyan, Lev A; Antonova, Irina B; Kuznetcov, Igor N; Gorbachev, Alexey Yu; Shakhparonov, Mikhail I; Govorun, Vadim M

2014-12-01

Ovarian cancer ascites is a native medium for cancer cells that allows investigation of their secretome in a natural environment. This medium is of interest as a promising source of potential biomarkers, and also as a medium for cell-cell communication. The aim of this study was to elucidate specific features of the malignant ascites metabolome and proteome. In order to omit components of the systemic response to ascites formation, we compared malignant ascites with cirrhosis ascites. Metabolome analysis revealed 41 components that differed significantly between malignant and cirrhosis ascites. Most of the identified cancer-specific metabolites are known to be important signaling molecules. Proteomic analysis identified 2096 and 1855 proteins in the ovarian cancer and cirrhosis ascites, respectively; 424 proteins were specific for the malignant ascites. Functional analysis of the proteome demonstrated that the major differences between cirrhosis and malignant ascites were observed for the cluster of spliceosomal proteins. Additionally, we demonstrate that several splicing RNAs were exclusively detected in malignant ascites, where they probably existed within protein complexes. This result was confirmed in vitro using an ovarian cancer cell line. Identification of spliceosomal proteins and RNAs in an extracellular medium is of particular interest; the finding suggests that they might play a role in the communication between cancer cells. In addition, malignant ascites contains a high number of exosomes that are known to play an important role in signal transduction. Thus our study reveals the specific features of malignant ascites that are associated with its function as a medium of intercellular communication. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Comparative and quantitative proteomics reveal the adaptive strategies of oyster larvae to ocean acidification

KAUST Repository

Dineshram, R.

2015-10-28

© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Decreasing pH due to anthropogenic CO2 inputs, called ocean acidification (OA), can make coastal environments unfavorable for oysters. This is a serious socioeconomical issue for China which supplies >70% of the world\\'s edible oysters. Here, we present an iTRAQ-based protein profiling approach for the detection and quantification of proteome changes under OA in the early life stage of a commercially important oyster, Crassostrea hongkongensis. Availability of complete genome sequence for the pacific oyster (Crassostrea gigas) enabled us to confidently quantify over 1500 proteins in larval oysters. Over 7% of the proteome was altered in response to OA at pHNBS 7.6. Analysis of differentially expressed proteins and their associated functional pathways showed an upregulation of proteins involved in calcification, metabolic processes, and oxidative stress, each of which may be important in physiological adaptation of this species to OA. The downregulation of cytoskeletal and signal transduction proteins, on the other hand, might have impaired cellular dynamics and organelle development under OA. However, there were no significant detrimental effects in developmental processes such as metamorphic success. Implications of the differentially expressed proteins and metabolic pathways in the development of OA resistance in oyster larvae are discussed. The MS proteomics data have been deposited to the ProteomeXchange with identifiers PXD002138 (http://proteomecentral.proteomexchange.org/dataset/PXD002138).

Comparative and quantitative proteomics reveal the adaptive strategies of oyster larvae to ocean acidification

KAUST Repository

Dineshram, R.; Q., Quan; Sharma, Rakesh; Chandramouli, Kondethimmanahalli; Yalamanchili, Hari Krishna; Chu, Ivan; Thiyagarajan, Vengatesen

2015-01-01

© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Decreasing pH due to anthropogenic CO2 inputs, called ocean acidification (OA), can make coastal environments unfavorable for oysters. This is a serious socioeconomical issue for China which supplies >70% of the world's edible oysters. Here, we present an iTRAQ-based protein profiling approach for the detection and quantification of proteome changes under OA in the early life stage of a commercially important oyster, Crassostrea hongkongensis. Availability of complete genome sequence for the pacific oyster (Crassostrea gigas) enabled us to confidently quantify over 1500 proteins in larval oysters. Over 7% of the proteome was altered in response to OA at pHNBS 7.6. Analysis of differentially expressed proteins and their associated functional pathways showed an upregulation of proteins involved in calcification, metabolic processes, and oxidative stress, each of which may be important in physiological adaptation of this species to OA. The downregulation of cytoskeletal and signal transduction proteins, on the other hand, might have impaired cellular dynamics and organelle development under OA. However, there were no significant detrimental effects in developmental processes such as metamorphic success. Implications of the differentially expressed proteins and metabolic pathways in the development of OA resistance in oyster larvae are discussed. The MS proteomics data have been deposited to the ProteomeXchange with identifiers PXD002138 (http://proteomecentral.proteomexchange.org/dataset/PXD002138).

The Skeleton Forming Proteome of an Early Branching Metazoan: A Molecular Survey of the Biomineralization Components Employed by the Coralline Sponge Vaceletia Sp.

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Juliane Germer

Full Text Available The ability to construct a mineralized skeleton was a major innovation for the Metazoa during their evolution in the late Precambrian/early Cambrian. Porifera (sponges hold an informative position for efforts aimed at unraveling the origins of this ability because they are widely regarded to be the earliest branching metazoans, and are among the first multi-cellular animals to display the ability to biomineralize in the fossil record. Very few biomineralization associated proteins have been identified in sponges so far, with no transcriptome or proteome scale surveys yet available. In order to understand what genetic repertoire may have been present in the last common ancestor of the Metazoa (LCAM, and that may have contributed to the evolution of the ability to biocalcify, we have studied the skeletal proteome of the coralline demosponge Vaceletia sp. and compare this to other metazoan biomineralizing proteomes. We bring some spatial resolution to this analysis by dividing Vaceletia's aragonitic calcium carbonate skeleton into "head" and "stalk" regions. With our approach we were able to identify 40 proteins from both the head and stalk regions, with many of these sharing some similarity to previously identified gene products from other organisms. Among these proteins are known biomineralization compounds, such as carbonic anhydrase, spherulin, extracellular matrix proteins and very acidic proteins. This report provides the first proteome scale analysis of a calcified poriferan skeletal proteome, and its composition clearly demonstrates that the LCAM contributed several key enzymes and matrix proteins to its descendants that supported the metazoan ability to biocalcify. However, lineage specific evolution is also likely to have contributed significantly to the ability of disparate metazoan lineages to biocalcify.

Establishing Substantial Equivalence: Proteomics

Science.gov (United States)

Lovegrove, Alison; Salt, Louise; Shewry, Peter R.

Wheat is a major crop in world agriculture and is consumed after processing into a range of food products. It is therefore of great importance to determine the consequences (intended and unintended) of transgenesis in wheat and whether genetically modified lines are substantially equivalent to those produced by conventional plant breeding. Proteomic analysis is one of several approaches which can be used to address these questions. Two-dimensional PAGE (2D PAGE) remains the most widely available method for proteomic analysis, but is notoriously difficult to reproduce between laboratories. We therefore describe methods which have been developed as standard operating procedures in our laboratory to ensure the reproducibility of proteomic analyses of wheat using 2D PAGE analysis of grain proteins.

Common proteomic changes in the hippocampus in schizophrenia and bipolar disorder and particular evidence for involvement of cornu ammonis regions 2 and 3.

LENUS (Irish Health Repository)

2011-05-01

The hippocampus is strongly implicated in schizophrenia and, to a lesser degree, bipolar disorder. Proteomic investigations of the different regions of the hippocampus may help us to clarify the basis and the disease specificity of the changes.

Novel asymmetrically localizing components of human centrosomes identified by complementary proteomics methods

DEFF Research Database (Denmark)

Jakobsen, Lis; Vanselow, Katja; Skogs, Marie

2011-01-01

by identifying a novel set of five proteins preferentially associated with mother or daughter centrioles, comprising genes implicated in cell polarity. Pulsed labelling demonstrates a remarkable variation in the stability of centrosomal protein complexes. These spatiotemporal proteomics data provide leads......Centrosomes in animal cells are dynamic organelles with a proteinaceous matrix of pericentriolar material assembled around a pair of centrioles. They organize the microtubule cytoskeleton and the mitotic spindle apparatus. Mature centrioles are essential for biogenesis of primary cilia that mediate...

Expression of small leucine-rich extracellular matrix proteoglycans biglycan and lumican reveals oral lichen planus malignant potential.

Science.gov (United States)

Lončar-Brzak, Božana; Klobučar, Marko; Veliki-Dalić, Irena; Sabol, Ivan; Kraljević Pavelić, Sandra; Krušlin, Božo; Mravak-Stipetić, Marinka

2018-03-01

The aim of this study was to examine molecular alterations on the protein level in lesions of oral lichen planus (OLP), oral squamous cell carcinoma (OSCC) and healthy mucosa. Global protein profiling methods based on liquid chromatography coupled to mass spectrometry (LC-MS) were used, with a special emphasis on evaluation of deregulated extracellular matrix molecules expression, as well as on analyses of IG2F and IGFR2 expression in healthy mucosa, OLP and OSCC tissues by comparative semi-quantitative immunohistochemistry. Mass spectrometry-based proteomics profiling of healthy mucosa, OLP and OSCC tissues (and accompanied histologically unaltered tissues, respectively) identified 55 extracellular matrix proteins. Twenty among identified proteins were common to all groups of samples. Expression of small leucine-rich extracellular matrix proteoglycans lumican and biglycan was found both in OSCC and OLP and they were validated by Western blot analysis as putative biomarkers. A significant increase (p < 0.05) of biglycan expression in OLP-AT group was determined in comparison with OLP-T group, while lumican showed significant up-regulation (p < 0.05) in OLP-T and OSCC-T groups vs. adjacent and control tissue groups. Biglycan expression was only determined in OSCC-AT group. Immunohistochemical analysis of IGF2 and IG2FR expression revealed no significant difference among groups of samples. Biglycan and lumican were identified as important pathogenesis biomarkers of OLP that point to its malignant potential.

Proteome identification of the silkworm middle silk gland

Directory of Open Access Journals (Sweden)

Jian-ying Li

2016-03-01

Full Text Available To investigate the functional differentiation among the anterior (A, middle (M, and posterior (P regions of silkworm middle silk gland (MSG, their proteomes were characterized by shotgun LC–MS/MS analysis with a LTQ-Orbitrap mass spectrometer. To get better proteome identification and quantification, triplicate replicates of mass spectrometry analysis were performed for each sample. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (Vizcaíno et al., 2014 [1] via the PRIDE partner repository (Vizcaino, 2013 [2] with the dataset identifier http://www.ebi.ac.uk/pride/archive/projects/PXD003371. The peptide identifications that were further processed by PeptideProphet program in Trans-Proteomic Pipeline (TPP after database search with Mascot software were also available in .XML format files. Data presented here are related to a research article published in Journal of Proteomics by Li et al. (2015 [3]. Keywords: Bombyx mori, Middle silk gland, Silk protein synthesis, Shotgun proteomics, Label-free

Genetic Background is a Key Determinant of Glomerular Extracellular Matrix Composition and Organization.

Science.gov (United States)

Randles, Michael J; Woolf, Adrian S; Huang, Jennifer L; Byron, Adam; Humphries, Jonathan D; Price, Karen L; Kolatsi-Joannou, Maria; Collinson, Sophie; Denny, Thomas; Knight, David; Mironov, Aleksandr; Starborg, Toby; Korstanje, Ron; Humphries, Martin J; Long, David A; Lennon, Rachel

2015-12-01

Glomerular disease often features altered histologic patterns of extracellular matrix (ECM). Despite this, the potential complexities of the glomerular ECM in both health and disease are poorly understood. To explore whether genetic background and sex determine glomerular ECM composition, we investigated two mouse strains, FVB and B6, using RNA microarrays of isolated glomeruli combined with proteomic glomerular ECM analyses. These studies, undertaken in healthy young adult animals, revealed unique strain- and sex-dependent glomerular ECM signatures, which correlated with variations in levels of albuminuria and known predisposition to progressive nephropathy. Among the variation, we observed changes in netrin 4, fibroblast growth factor 2, tenascin C, collagen 1, meprin 1-α, and meprin 1-β. Differences in protein abundance were validated by quantitative immunohistochemistry and Western blot analysis, and the collective differences were not explained by mutations in known ECM or glomerular disease genes. Within the distinct signatures, we discovered a core set of structural ECM proteins that form multiple protein-protein interactions and are conserved from mouse to man. Furthermore, we found striking ultrastructural changes in glomerular basement membranes in FVB mice. Pathway analysis of merged transcriptomic and proteomic datasets identified potential ECM regulatory pathways involving inhibition of matrix metalloproteases, liver X receptor/retinoid X receptor, nuclear factor erythroid 2-related factor 2, notch, and cyclin-dependent kinase 5. These pathways may therefore alter ECM and confer susceptibility to disease. Copyright © 2015 by the American Society of Nephrology.

Hospitalized Premature Infants Are Colonized by Related Bacterial Strains with Distinct Proteomic Profiles

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Christopher T. Brown

2018-04-01

Full Text Available During the first weeks of life, microbial colonization of the gut impacts human immune system maturation and other developmental processes. In premature infants, aberrant colonization has been implicated in the onset of necrotizing enterocolitis (NEC, a life-threatening intestinal disease. To study the premature infant gut colonization process, genome-resolved metagenomics was conducted on 343 fecal samples collected during the first 3 months of life from 35 premature infants housed in a neonatal intensive care unit, 14 of whom developed NEC, and metaproteomic measurements were made on 87 samples. Microbial community composition and proteomic profiles remained relatively stable on the time scale of a week, but the proteome was more variable. Although genetically similar organisms colonized many infants, most infants were colonized by distinct strains with metabolic profiles that could be distinguished using metaproteomics. Microbiome composition correlated with infant, antibiotics administration, and NEC diagnosis. Communities were found to cluster into seven primary types, and community type switched within infants, sometimes multiple times. Interestingly, some communities sampled from the same infant at subsequent time points clustered with those of other infants. In some cases, switches preceded onset of NEC; however, no species or community type could account for NEC across the majority of infants. In addition to a correlation of protein abundances with organism replication rates, we found that organism proteomes correlated with overall community composition. Thus, this genome-resolved proteomics study demonstrated that the contributions of individual organisms to microbiome development depend on microbial community context.

Hospitalized Premature Infants Are Colonized by Related Bacterial Strains with Distinct Proteomic Profiles

Science.gov (United States)

Xiong, Weili; Olm, Matthew R.; Thomas, Brian C.; Baker, Robyn; Firek, Brian; Morowitz, Michael J.; Hettich, Robert L.

2018-01-01

ABSTRACT During the first weeks of life, microbial colonization of the gut impacts human immune system maturation and other developmental processes. In premature infants, aberrant colonization has been implicated in the onset of necrotizing enterocolitis (NEC), a life-threatening intestinal disease. To study the premature infant gut colonization process, genome-resolved metagenomics was conducted on 343 fecal samples collected during the first 3 months of life from 35 premature infants housed in a neonatal intensive care unit, 14 of whom developed NEC, and metaproteomic measurements were made on 87 samples. Microbial community composition and proteomic profiles remained relatively stable on the time scale of a week, but the proteome was more variable. Although genetically similar organisms colonized many infants, most infants were colonized by distinct strains with metabolic profiles that could be distinguished using metaproteomics. Microbiome composition correlated with infant, antibiotics administration, and NEC diagnosis. Communities were found to cluster into seven primary types, and community type switched within infants, sometimes multiple times. Interestingly, some communities sampled from the same infant at subsequent time points clustered with those of other infants. In some cases, switches preceded onset of NEC; however, no species or community type could account for NEC across the majority of infants. In addition to a correlation of protein abundances with organism replication rates, we found that organism proteomes correlated with overall community composition. Thus, this genome-resolved proteomics study demonstrated that the contributions of individual organisms to microbiome development depend on microbial community context. PMID:29636439

A comprehensive proteomics study on platelet concentrates: Platelet proteome, storage time and Mirasol pathogen reduction technology.

Science.gov (United States)

Salunkhe, Vishal; De Cuyper, Iris M; Papadopoulos, Petros; van der Meer, Pieter F; Daal, Brunette B; Villa-Fajardo, María; de Korte, Dirk; van den Berg, Timo K; Gutiérrez, Laura

2018-03-19

Platelet concentrates (PCs) represent a blood transfusion product with a major concern for safety as their storage temperature (20-24°C) allows bacterial growth, and their maximum storage time period (less than a week) precludes complete microbiological testing. Pathogen inactivation technologies (PITs) provide an additional layer of safety to the blood transfusion products from known and unknown pathogens such as bacteria, viruses, and parasites. In this context, PITs, such as Mirasol Pathogen Reduction Technology (PRT), have been developed and are implemented in many countries. However, several studies have shown in vitro that Mirasol PRT induces a certain level of platelet shape change, hyperactivation, basal degranulation, and increased oxidative damage during storage. It has been suggested that Mirasol PRT might accelerate what has been described as the platelet storage lesion (PSL), but supportive molecular signatures have not been obtained. We aimed at dissecting the influence of both variables, that is, Mirasol PRT and storage time, at the proteome level. We present comprehensive proteomics data analysis of Control PCs and PCs treated with Mirasol PRT at storage days 1, 2, 6, and 8. Our workflow was set to perform proteomics analysis using a gel-free and label-free quantification (LFQ) approach. Semi-quantification was based on LFQ signal intensities of identified proteins using MaxQuant/Perseus software platform. Data are available via ProteomeXchange with identifier PXD008119. We identified marginal differences between Mirasol PRT and Control PCs during storage. However, those significant changes at the proteome level were specifically related to the functional aspects previously described to affect platelets upon Mirasol PRT. In addition, the effect of Mirasol PRT on the platelet proteome appeared not to be exclusively due to an accelerated or enhanced PSL. In summary, semi-quantitative proteomics allows to discern between proteome changes due to

Proteomic profiling identifies markers for inflammation-related tumor-fibroblast interaction.

Science.gov (United States)

Drev, Daniel; Bileck, Andrea; Erdem, Zeynep N; Mohr, Thomas; Timelthaler, Gerald; Beer, Andrea; Gerner, Christopher; Marian, Brigitte

2017-01-01

Cancer associated fibroblasts are activated in the tumor microenvironment and contribute to tumor progression, angiogenesis, extracellular matrix remodeling, and inflammation. To identify proteins characteristic for fibroblasts in colorectal cancer we used liquid chromatography-tandem mass spectrometry to derive protein abundance from whole-tissue homogenates of human colorectal cancer/normal mucosa pairs. Alterations of protein levels were determined by two-sided t test with greater than threefold difference and an FDR of matrix organization, TGFβ receptor signaling and angiogenesis mainly originating from the stroma. Most prominent were increased abundance of SerpinB5 in the parenchyme and latent transforming growth factor β-binding protein, thrombospondin-B2, and secreted protein acidic-and-cysteine-rich in the stroma. Extracellular matrix remodeling involved collagens type VIII, XII, XIV, and VI as well as lysyl-oxidase-2. In silico analysis of mRNA levels demonstrated altered expression in the tumor and the adjacent normal tissue as compared to mucosa of healthy individuals indicating that inflammatory activation affected the surrounding tissue. Immunohistochemistry of 26 tumor specimen confirmed upregulation of SerpinB5, thrombospondin B2 and secreted protein acidic-and-cysteine-rich. This study demonstrates the feasibility of detecting tumor- and compartment-specific protein-signatures that are functionally meaningful by proteomic profiling of whole-tissue extracts together with mining of RNA expression datasets. The results provide the basis for further exploration of inflammation-related stromal markers in larger patient cohorts and experimental models.

Proteome-wide analysis and diel proteomic profiling of the cyanobacterium Arthrospira platensis PCC 8005.

Directory of Open Access Journals (Sweden)

Sabine Matallana-Surget

Full Text Available The filamentous cyanobacterium Arthrospira platensis has a long history of use as a food supply and it has been used by the European Space Agency in the MELiSSA project, an artificial microecosystem which supports life during long-term manned space missions. This study assesses progress in the field of cyanobacterial shotgun proteomics and light/dark diurnal cycles by focusing on Arthrospira platensis. Several fractionation workflows including gel-free and gel-based protein/peptide fractionation procedures were used and combined with LC-MS/MS analysis, enabling the overall identification of 1306 proteins, which represents 21% coverage of the theoretical proteome. A total of 30 proteins were found to be significantly differentially regulated under light/dark growth transition. Interestingly, most of the proteins showing differential abundance were related to photosynthesis, the Calvin cycle and translation processes. A novel aspect and major achievement of this work is the successful improvement of the cyanobacterial proteome coverage using a 3D LC-MS/MS approach, based on an immobilized metal affinity chromatography, a suitable tool that enabled us to eliminate the most abundant protein, the allophycocyanin. We also demonstrated that cell growth follows a light/dark cycle in A. platensis. This preliminary proteomic study has highlighted new characteristics of the Arthrospira platensis proteome in terms of diurnal regulation.

Comparison of protein extraction methods suitable for proteomics ...

African Journals Online (AJOL)

Jane

2011-07-27

Jul 27, 2011 ... An efficient protein extraction method is a prerequisite for successful implementation of proteomics. ... research, it is noteworthy to discover a proteome ..... Proteomic analysis of rice (Oryza sativa) seeds during germination.

Extracellular vesicles in gastrointestinal cancer in conjunction with microbiota: On the border of Kingdoms

KAUST Repository

Barteneva, Natasha S.

2017-06-29

Extracellular vesicle (EV) production is a universal feature of metazoan cells as well as prokaryotes (bMVs - bacterial microvesicls). They are small vesicles with phospholipid membrane carrying proteins, DNA and different classes of RNAs and are heavily involved in intercellular communication acting as vectors of information to target cells. For the last decade, the interest in EV research has exponentially increased though thorough studies of their roles in various pathologies that was not previously possible due to technical limitations.This review focuses on research evaluating the role of EV production in gastrointestinal (GI) cancer development in conjunction with GI microbiota and inflammatory diseases. We also discuss recent studies on the promising role of EVs and their content as biomarkers for early diagnosis of GI cancers. The bMVs have also been implicated in the pathogenesis of GI chronic inflammatory diseases, however, possible role of bMVs in tumorigenesis remains underestimated. We propose that EVs from eukaryotic cells as well as from different microbial, fungi, parasitic species and edible plants in GI tract act as mediators of intracellular and inter-species communication, particularly facilitating tumour cell survival and multi-drug resistance. In conclusion, we suggest that matching sequences from EV proteomes (available from public databases) with known protein sequences of microbiome gut bacteria will be useful in identification of antigen mimicry between evolutionary conservative protein sequences. Using this approach we identified Bacteroides spp. pseudokinase with activation loop and homology to PDGFRα, providing a proof-of-concept strategy. We speculate that existence of microbial pseudokinase that ‘mimic” PDGFRα may be related to PDGFRα and Bacteroides spp. roles in colorectal carcinogenesis that require further investigation.

Urine Proteomics in the Era of Mass Spectrometry

Directory of Open Access Journals (Sweden)

Ashley Beasley-Green

2016-11-01

Full Text Available With the technological advances of mass spectrometry (MS-based platforms, clinical proteomics is one of the most rapidly growing areas in biomedical research. Urine proteomics has become a popular subdiscipline of clinical proteomics because it is an ideal source for the discovery of noninvasive disease biomarkers. The urine proteome offers a comprehensive view of the local and systemic physiology since the proteome is primarily composed of proteins/peptides from the kidneys and plasma. The emergence of MS-based proteomic platforms as prominent bioanalytical tools in clinical applications has enhanced the identification of protein-based urinary biomarkers. This review highlights the characteristics of urine that make it an attractive biofluid for biomarker discovery and the impact of MS-based technologies on the clinical assessment of urinary protein biomarkers.

Birth of plant proteomics in India: a new horizon.

Science.gov (United States)

Narula, Kanika; Pandey, Aarti; Gayali, Saurabh; Chakraborty, Niranjan; Chakraborty, Subhra

2015-09-08

In the post-genomic era, proteomics is acknowledged as the next frontier for biological research. Although India has a long and distinguished tradition in protein research, the initiation of proteomics studies was a new horizon. Protein research witnessed enormous progress in protein separation, high-resolution refinements, biochemical identification of the proteins, protein-protein interaction, and structure-function analysis. Plant proteomics research, in India, began its journey on investigation of the proteome profiling, complexity analysis, protein trafficking, and biochemical modeling. The research article by Bhushan et al. in 2006 marked the birth of the plant proteomics research in India. Since then plant proteomics studies expanded progressively and are now being carried out in various institutions spread across the country. The compilation presented here seeks to trace the history of development in the area during the past decade based on publications till date. In this review, we emphasize on outcomes of the field providing prospects on proteomic pathway analyses. Finally, we discuss the connotation of strategies and the potential that would provide the framework of plant proteome research. The past decades have seen rapidly growing number of sequenced plant genomes and associated genomic resources. To keep pace with this increasing body of data, India is in the provisional phase of proteomics research to develop a comparative hub for plant proteomes and protein families, but it requires a strong impetus from intellectuals, entrepreneurs, and government agencies. Here, we aim to provide an overview of past, present and future of Indian plant proteomics, which would serve as an evaluation platform for those seeking to incorporate proteomics into their research programs. This article is part of a Special Issue entitled: Proteomics in India. Copyright © 2015 Elsevier B.V. All rights reserved.

The Redox Proteome*

Science.gov (United States)

Go, Young-Mi; Jones, Dean P.

2013-01-01

The redox proteome consists of reversible and irreversible covalent modifications that link redox metabolism to biologic structure and function. These modifications, especially of Cys, function at the molecular level in protein folding and maturation, catalytic activity, signaling, and macromolecular interactions and at the macroscopic level in control of secretion and cell shape. Interaction of the redox proteome with redox-active chemicals is central to macromolecular structure, regulation, and signaling during the life cycle and has a central role in the tolerance and adaptability to diet and environmental challenges. PMID:23861437

Global Proteome Response to Deletion of Genes Related to Mercury Methylation and Dissimilatory Metal Reduction Reveals Changes in Respiratory Metabolism in Geobacter sulfurreducens PCA.

Science.gov (United States)

Qian, Chen; Johs, Alexander; Chen, Hongmei; Mann, Benjamin F; Lu, Xia; Abraham, Paul E; Hettich, Robert L; Gu, Baohua

2016-10-07

Geobacter sulfurreducens PCA can reduce, sorb, and methylate mercury (Hg); however, the underlying biochemical mechanisms of these processes and interdependent metabolic pathways remain unknown. In this study, shotgun proteomics was used to compare global proteome profiles between wild-type G. sulfurreducens PCA and two mutant strains: a ΔhgcAB mutant, which is deficient in two genes known to be essential for Hg methylation and a ΔomcBESTZ mutant, which is deficient in five outer membrane c-type cytochromes and thus impaired in its ability for dissimilatory metal ion reduction. We were able to delineate the global response of G. sulfurreducens PCA in both mutants and identify cellular networks and metabolic pathways that were affected by the loss of these genes. Deletion of hgcAB increased the relative abundances of proteins implicated in extracellular electron transfer, including most of the c-type cytochromes, PilA-C, and OmpB, and is consistent with a previously observed increase in Hg reduction in the ΔhgcAB mutant. Deletion of omcBESTZ was found to significantly increase relative abundances of various methyltransferases, suggesting that a loss of dissimilatory reduction capacity results in elevated activity among one-carbon (C1) metabolic pathways and thus increased methylation. We show that G. sulfurreducens PCA encodes only the folate branch of the acetyl-CoA pathway, and proteins associated with the folate branch were found at lower abundance in the ΔhgcAB mutant strain than the wild type. This observation supports the hypothesis that the function of HgcA and HgcB is linked to C1 metabolism through the folate branch of the acetyl-CoA pathway by providing methyl groups required for Hg methylation.

Proteomic landscape in Central and Eastern Europe: the 9th Central and Eastern European Proteomic Conference, Poznań, Poland.

Science.gov (United States)

Gadher, Suresh Jivan; Marczak, Łukasz; Łuczak, Magdalena; Stobiecki, Maciej; Widlak, Piotr; Kovarova, Hana

2016-01-01

Every year since 2007, the Central and Eastern European Proteomic Conference (CEEPC) has excelled in representing state-of-the-art proteomics in and around Central and Eastern Europe, and linking it to international institutions worldwide. Its mission remains to contribute to all approaches of proteomics including traditional and often-revisited methodologies as well as the latest technological achievements in clinical, quantitative and structural proteomics with a view to systems biology of a variety of processes. The 9th CEEPC was held from June 15th to 18th, 2015, at the Institute of Bioorganic Chemistry, Polish Academy of Sciences in Poznań, Poland. The scientific program stimulated exchange of proteomic knowledge whilst the spectacular venue of the conference allowed participants to enjoy the cobblestoned historical city of Poznań.

Application of proteomics to ecology and population biology.

Science.gov (United States)

Karr, T L

2008-02-01

Proteomics is a relatively new scientific discipline that merges protein biochemistry, genome biology and bioinformatics to determine the spatial and temporal expression of proteins in cells, tissues and whole organisms. There has been very little application of proteomics to the fields of behavioral genetics, evolution, ecology and population dynamics, and has only recently been effectively applied to the closely allied fields of molecular evolution and genetics. However, there exists considerable potential for proteomics to impact in areas related to functional ecology; this review will introduce the general concepts and methodologies that define the field of proteomics and compare and contrast the advantages and disadvantages with other methods. Examples of how proteomics can aid, complement and indeed extend the study of functional ecology will be discussed including the main tool of ecological studies, population genetics with an emphasis on metapopulation structure analysis. Because proteomic analyses provide a direct measure of gene expression, it obviates some of the limitations associated with other genomic approaches, such as microarray and EST analyses. Likewise, in conjunction with associated bioinformatics and molecular evolutionary tools, proteomics can provide the foundation of a systems-level integration approach that can enhance ecological studies. It can be envisioned that proteomics will provide important new information on issues specific to metapopulation biology and adaptive processes in nature. A specific example of the application of proteomics to sperm ageing is provided to illustrate the potential utility of the approach.

Virtual Labs in proteomics: new E-learning tools.

Science.gov (United States)

Ray, Sandipan; Koshy, Nicole Rachel; Reddy, Panga Jaipal; Srivastava, Sanjeeva

2012-05-17

Web-based educational resources have gained enormous popularity recently and are increasingly becoming a part of modern educational systems. Virtual Labs are E-learning platforms where learners can gain the experience of practical experimentation without any direct physical involvement on real bench work. They use computerized simulations, models, videos, animations and other instructional technologies to create interactive content. Proteomics being one of the most rapidly growing fields of the biological sciences is now an important part of college and university curriculums. Consequently, many E-learning programs have started incorporating the theoretical and practical aspects of different proteomic techniques as an element of their course work in the form of Video Lectures and Virtual Labs. To this end, recently we have developed a Virtual Proteomics Lab at the Indian Institute of Technology Bombay, which demonstrates different proteomics techniques, including basic and advanced gel and MS-based protein separation and identification techniques, bioinformatics tools and molecular docking methods, and their applications in different biological samples. This Tutorial will discuss the prominent Virtual Labs featuring proteomics content, including the Virtual Proteomics Lab of IIT-Bombay, and E-resources available for proteomics study that are striving to make proteomic techniques and concepts available and accessible to the student and research community. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP 14). Details can be found at: http://www.proteomicstutorials.org/. Copyright © 2012 Elsevier B.V. All rights reserved.

Analysis of mass spectrometry data in proteomics

DEFF Research Database (Denmark)

Matthiesen, Rune; Jensen, Ole N

2008-01-01

The systematic study of proteins and protein networks, that is, proteomics, calls for qualitative and quantitative analysis of proteins and peptides. Mass spectrometry (MS) is a key analytical technology in current proteomics and modern mass spectrometers generate large amounts of high-quality data...... that in turn allow protein identification, annotation of secondary modifications, and determination of the absolute or relative abundance of individual proteins. Advances in mass spectrometry-driven proteomics rely on robust bioinformatics tools that enable large-scale data analysis. This chapter describes...... some of the basic concepts and current approaches to the analysis of MS and MS/MS data in proteomics....

Maillard Proteomics: Opening New Pages

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Alena Soboleva

2017-12-01

Full Text Available Protein glycation is a ubiquitous non-enzymatic post-translational modification, formed by reaction of protein amino and guanidino groups with carbonyl compounds, presumably reducing sugars and α-dicarbonyls. Resulting advanced glycation end products (AGEs represent a highly heterogeneous group of compounds, deleterious in mammals due to their pro-inflammatory effect, and impact in pathogenesis of diabetes mellitus, Alzheimer’s disease and ageing. The body of information on the mechanisms and pathways of AGE formation, acquired during the last decades, clearly indicates a certain site-specificity of glycation. It makes characterization of individual glycation sites a critical pre-requisite for understanding in vivo mechanisms of AGE formation and developing adequate nutritional and therapeutic approaches to reduce it in humans. In this context, proteomics is the methodology of choice to address site-specific molecular changes related to protein glycation. Therefore, here we summarize the methods of Maillard proteomics, specifically focusing on the techniques providing comprehensive structural and quantitative characterization of glycated proteome. Further, we address the novel break-through areas, recently established in the field of Maillard research, i.e., in vitro models based on synthetic peptides, site-based diagnostics of metabolism-related diseases (e.g., diabetes mellitus, proteomics of anti-glycative defense, and dynamics of plant glycated proteome during ageing and response to environmental stress.

Knowledge Translation: Moving Proteomics Science to Innovation in Society.

Science.gov (United States)

Holmes, Christina; McDonald, Fiona; Jones, Mavis; Graham, Janice

2016-06-01

Proteomics is one of the pivotal next-generation biotechnologies in the current "postgenomics" era. Little is known about the ways in which innovative proteomics science is navigating the complex socio-political space between laboratory and society. It cannot be assumed that the trajectory between proteomics laboratory and society is linear and unidirectional. Concerned about public accountability and hopes for knowledge-based innovations, funding agencies and citizens increasingly expect that emerging science and technologies, such as proteomics, are effectively translated and disseminated as innovation in society. Here, we describe translation strategies promoted in the knowledge translation (KT) and science communication literatures and examine the use of these strategies within the field of proteomics. Drawing on data generated from qualitative interviews with proteomics scientists and ethnographic observation of international proteomics conferences over a 5-year period, we found that proteomics science incorporates a variety of KT strategies to reach knowledge users outside the field. To attain the full benefit of KT, however, proteomics scientists must challenge their own normative assumptions and approaches to innovation dissemination-beyond the current paradigm relying primarily on publication for one's scientific peers within one's field-and embrace the value of broader (interdisciplinary) KT strategies in promoting the uptake of their research. Notably, the Human Proteome Organization (HUPO) is paying increasing attention to a broader range of KT strategies, including targeted dissemination, integrated KT, and public outreach. We suggest that increasing the variety of KT strategies employed by proteomics scientists is timely and would serve well the omics system sciences community.

First systematic plant proteomics workshop in Botany Department, University of Delhi: transferring proteomics knowledge to next-generation researchers and students.

Science.gov (United States)

Deswal, Renu; Abat, Jasmeet Kaur; Sehrawat, Ankita; Gupta, Ravi; Kashyap, Prakriti; Sharma, Shruti; Sharma, Bhavana; Chaurasia, Satya Prakash; Chanu, Sougrakpam Yaiphabi; Masi, Antonio; Agrawal, Ganesh Kumar; Sarkar, Abhijit; Agrawal, Raj; Dunn, Michael J; Renaut, Jenny; Rakwal, Randeep

2014-07-01

International Plant Proteomics Organization (INPPO) outlined ten initiatives to promote plant proteomics in each and every country. With greater emphasis in developing countries, one of those was to "organize workshops at national and international levels to train manpower and exchange information". This third INPPO highlights covers the workshop organized for the very first time in a developing country, India, at the Department of Botany in University of Delhi on December 26-30, 2013 titled - "1(st) Plant Proteomics Workshop / Training Program" under the umbrella of INPPO India-Nepal chapter. Selected 20 participants received on-hand training mainly on gel-based proteomics approach along with manual booklet and parallel lectures on this and associated topics. In house, as well as invited experts drawn from other Universities and Institutes (national and international), delivered talks on different aspects of gel-based and gel-free proteomics. Importance of gel-free proteomics approach, translational proteomics, and INPPO roles were presented and interactively discussed by a group of three invited speakers Drs. Ganesh Kumar Agrawal (Nepal), Randeep Rakwal (Japan), and Antonio Masi (Italy). Given the output of this systematic workshop, it was proposed and thereafter decided to be organized every alternate year; the next workshop will be held in 2015. Furthermore, possibilities on providing advanced training to those students / researchers / teachers with basic knowledge in proteomics theory and experiments at national and international levels were discussed. INPPO is committed to generating next-generation trained manpower in proteomics, and it would only happen by the firm determination of scientists to come forward and do it. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Diversity of extracellular proteins during the transition from the ‘proto-apicomplexan’ alveolates to the apicomplexan obligate parasites

KAUST Repository

Templeton, Thomas J.; Pain, Arnab

2015-01-01

The recent completion of high-coverage draft genome sequences for several alveolate protozoans – namely, the chromerids, Chromera velia and Vitrella brassicaformis ; the perkinsid Perkinsus marinus ; the apicomplexan, Gregarina niphandrodes , as well as high coverage transcriptome sequence information for several colpodellids, allows for new genome-scale comparisons across a rich landscape of apicomplexans and other alveolates. Genome annotations can now be used to help interpret fine ultrastructure and cell biology, and guide new studies to describe a variety of alveolate life strategies, such as symbiosis or free living, predation, and obligate intracellular parasitism, as well to provide foundations to dissect the evolutionary transitions between these niches. This review focuses on the attempt to identify extracellular proteins which might mediate the physical interface of cell–cell interactions within the above life strategies, aided by annotation of the repertoires of predicted surface and secreted proteins encoded within alveolate genomes. In particular, we discuss what descriptions of the predicted extracellular proteomes reveal regarding a hypothetical last common ancestor of a pre-apicomplexan alveolate – guided by ultrastructure, life strategies and phylogenetic relationships – in an attempt to understand the evolution of obligate parasitism in apicomplexans.

Diversity of extracellular proteins during the transition from the ‘proto-apicomplexan’ alveolates to the apicomplexan obligate parasites

KAUST Repository

Templeton, Thomas J.

2015-11-20

The recent completion of high-coverage draft genome sequences for several alveolate protozoans – namely, the chromerids, Chromera velia and Vitrella brassicaformis ; the perkinsid Perkinsus marinus ; the apicomplexan, Gregarina niphandrodes , as well as high coverage transcriptome sequence information for several colpodellids, allows for new genome-scale comparisons across a rich landscape of apicomplexans and other alveolates. Genome annotations can now be used to help interpret fine ultrastructure and cell biology, and guide new studies to describe a variety of alveolate life strategies, such as symbiosis or free living, predation, and obligate intracellular parasitism, as well to provide foundations to dissect the evolutionary transitions between these niches. This review focuses on the attempt to identify extracellular proteins which might mediate the physical interface of cell–cell interactions within the above life strategies, aided by annotation of the repertoires of predicted surface and secreted proteins encoded within alveolate genomes. In particular, we discuss what descriptions of the predicted extracellular proteomes reveal regarding a hypothetical last common ancestor of a pre-apicomplexan alveolate – guided by ultrastructure, life strategies and phylogenetic relationships – in an attempt to understand the evolution of obligate parasitism in apicomplexans.

Thermally assisted acoustofluidic separation of extracellular vesicles from cells

Science.gov (United States)

Mirtaheri, Elnaz; Dolatmoradi, Ata; Pimentel, Krystine; Bhansali, Shekhar; El-Zahab, Bilal

2018-02-01

Extracellular vesicles (EVs) have been gaining increasing attention given their role in communicating information between cells. Composition-based isolation of EVs is particularly of high significance as the proteomic and lipidomic characterization of their cargo could provide valuable clues to the role of EVs in mediating the biology of various conditions. This has, however, proved to be challenging as EVs, despite their abundance, are very small and difficult to be differentiated from the other constituents of host media. In addition, currently available methods like ultracentrifugation and filtration are cumbersome and capable of achieving mostly size-based separations. In this work, we demonstrate the possibility of separating submicron EV-like vesicles from cancer cells using a thermally-assisted acoustophoretic device. In a system composed of MCF-7 breast cancer cells spiked with two different types of same-size vesicles, composition-based isolation of vesicles was shown to be realizable through opposite focusing of the system's components at the node and antinodes of the overlaid ultrasonic standing wave. By proper choice of temperature in the microchannel, we were able to achieve separations with purities exceeding 93%. Furthermore, cells recovered from the channel were shown to be viable after the separation.

Time- and radiation-dose dependent changes in the plasma proteome after total body irradiation of non-human primates: Implications for biomarker selection.

Directory of Open Access Journals (Sweden)

Stephanie D Byrum

Full Text Available Acute radiation syndrome (ARS is a complex multi-organ disease resulting from total body exposure to high doses of radiation. Individuals can be exposed to total body irradiation (TBI in a number of ways, including terrorist radiological weapons or nuclear accidents. In order to determine whether an individual has been exposed to high doses of radiation and needs countermeasure treatment, robust biomarkers are needed to estimate radiation exposure from biospecimens such as blood or urine. In order to identity such candidate biomarkers of radiation exposure, high-resolution proteomics was used to analyze plasma from non-human primates following whole body irradiation (Co-60 at 6.7 Gy and 7.4 Gy with a twelve day observation period. A total of 663 proteins were evaluated from the plasma proteome analysis. A panel of plasma proteins with characteristic time- and dose-dependent changes was identified. In addition to the plasma proteomics study reported here, we recently identified candidate biomarkers using urine from these same non-human primates. From the proteomic analysis of both plasma and urine, we identified ten overlapping proteins that significantly differentiate both time and dose variables. These shared plasma and urine proteins represent optimal candidate biomarkers of radiation exposure.

Proteomics methods applied to malaria: Plasmodium falciparum

International Nuclear Information System (INIS)

Cuesta Astroz, Yesid; Segura Latorre, Cesar

2012-01-01

Malaria is a parasitic disease that has a high impact on public health in developing countries. The sequencing of the plasmodium falciparum genome and the development of proteomics have enabled a breakthrough in understanding the biology of the parasite. Proteomics have allowed to characterize qualitatively and quantitatively the parasite s expression of proteins and has provided information on protein expression under conditions of stress induced by antimalarial. Given the complexity of their life cycle, this takes place in the vertebrate host and mosquito vector. It has proven difficult to characterize the protein expression during each stage throughout the infection process in order to determine the proteome that mediates several metabolic, physiological and energetic processes. Two dimensional electrophoresis, liquid chromatography and mass spectrometry have been useful to assess the effects of antimalarial on parasite protein expression and to characterize the proteomic profile of different p. falciparum stages and organelles. The purpose of this review is to present state of the art tools and advances in proteomics applied to the study of malaria, and to present different experimental strategies used to study the parasite's proteome in order to show the advantages and disadvantages of each one.

Scrambled eggs: Proteomic portraits and novel biomarkers of egg quality in zebrafish (Danio rerio.

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Ozlem Yilmaz

Full Text Available Egg quality is a complex biological trait and a major determinant of reproductive fitness in all animals. This study delivered the first proteomic portraits of egg quality in zebrafish, a leading biomedical model for early development. Egg batches of good and poor quality, evidenced by embryo survival for 24 h, were sampled immediately after spawning and used to create pooled or replicated sample sets whose protein extracts were subjected to different levels of fractionation before liquid chromatography and tandem mass spectrometry. Obtained spectra were searched against a zebrafish proteome database and detected proteins were annotated, categorized and quantified based on normalized spectral counts. Manually curated and automated enrichment analyses revealed poor quality eggs to be deficient of proteins involved in protein synthesis and energy and lipid metabolism, and of some vitellogenin products and lectins, and to have a surfeit of proteins involved in endo-lysosomal activities, autophagy, and apoptosis, and of some oncogene products, lectins and egg envelope proteins. Results of pathway and network analyses suggest that this aberrant proteomic profile results from failure of oocytes giving rise to poor quality eggs to properly transit through final maturation, and implicated Wnt signaling in the etiology of this defect. Quantitative comparisons of abundant proteins in good versus poor quality eggs revealed 17 candidate egg quality markers. Thus, the zebrafish egg proteome is clearly linked to embryo developmental potential, a phenomenon that begs further investigation to elucidate the root causes of poor egg quality, presently a serious and intractable problem in livestock and human reproductive medicine.

Scrambled eggs: Proteomic portraits and novel biomarkers of egg quality in zebrafish (Danio rerio).

Science.gov (United States)

Yilmaz, Ozlem; Patinote, Amélie; Nguyen, Thao Vi; Com, Emmanuelle; Lavigne, Regis; Pineau, Charles; Sullivan, Craig V; Bobe, Julien

2017-01-01

Egg quality is a complex biological trait and a major determinant of reproductive fitness in all animals. This study delivered the first proteomic portraits of egg quality in zebrafish, a leading biomedical model for early development. Egg batches of good and poor quality, evidenced by embryo survival for 24 h, were sampled immediately after spawning and used to create pooled or replicated sample sets whose protein extracts were subjected to different levels of fractionation before liquid chromatography and tandem mass spectrometry. Obtained spectra were searched against a zebrafish proteome database and detected proteins were annotated, categorized and quantified based on normalized spectral counts. Manually curated and automated enrichment analyses revealed poor quality eggs to be deficient of proteins involved in protein synthesis and energy and lipid metabolism, and of some vitellogenin products and lectins, and to have a surfeit of proteins involved in endo-lysosomal activities, autophagy, and apoptosis, and of some oncogene products, lectins and egg envelope proteins. Results of pathway and network analyses suggest that this aberrant proteomic profile results from failure of oocytes giving rise to poor quality eggs to properly transit through final maturation, and implicated Wnt signaling in the etiology of this defect. Quantitative comparisons of abundant proteins in good versus poor quality eggs revealed 17 candidate egg quality markers. Thus, the zebrafish egg proteome is clearly linked to embryo developmental potential, a phenomenon that begs further investigation to elucidate the root causes of poor egg quality, presently a serious and intractable problem in livestock and human reproductive medicine.

The Seed Proteome Web Portal

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Marc eGalland

2012-06-01

Full Text Available The Seed Proteome Web Portal (SPWP; http://www.seedproteome.com/ gives access to information both on quantitative seed proteomic data and on seed-related protocols. Firstly, the SPWP provides access to the 475 different Arabidopsis seed proteins annotated from 2 dimensional electrophoresis (2DE maps. Quantitative data are available for each protein according to their accumulation profile during the germination process. These proteins can be retrieved either in list format or directly on scanned 2DE maps. These proteomic data reveal that 40% of seed proteins maintain a stable abundance over germination, up to radicle protrusion. During sensu stricto germination (24 h upon imbibition about 50% of the proteins display quantitative variations, exhibiting an increased abundance (35% or a decreasing abundance (15%. Moreover, during radicle protrusion (24 h to 48 h upon imbibition, 41% proteins display quantitative variations with an increased (23% or a decreasing abundance (18%. In addition, an analysis of the seed proteome revealed the importance of protein post-translational modifications as demonstrated by the poor correlation (r2 = 0.29 between the theoretical (predicted from Arabidopsis genome and the observed protein isoelectric points. Secondly, the SPWP is a relevant technical resource for protocols specifically dedicated to Arabidopsis seed proteome studies. Concerning 2D electrophoresis, the user can find efficient procedures for sample preparation, electrophoresis coupled with gel analysis and protein identification by mass spectrometry, which we have routinely used during the last 12 years. Particular applications such as the detection of oxidized proteins or de novo synthetized proteins radiolabeled by [35S]-methionine are also given in great details. Future developments of this portal will include proteomic data from studies such as dormancy release and protein turnover through de novo protein synthesis analyses during germination.

Dentistry proteomics: from laboratory development to clinical practice.

Science.gov (United States)

Rezende, Taia M B; Lima, Stella M F; Petriz, Bernardo A; Silva, Osmar N; Freire, Mirna S; Franco, Octávio L

2013-12-01

Despite all the dental information acquired over centuries and the importance of proteome research, the cross-link between these two areas only emerged around mid-nineties. Proteomic tools can help dentistry in the identification of risk factors, early diagnosis, prevention, and systematic control that will promote the evolution of treatment in all dentistry specialties. This review mainly focuses on the evolution of dentistry in different specialties based on proteomic research and how these tools can improve knowledge in dentistry. The subjects covered are an overview of proteomics in dentistry, specific information on different fields in dentistry (dental structure, restorative dentistry, endodontics, periodontics, oral pathology, oral surgery, and orthodontics) and future directions. There are many new proteomic technologies that have never been used in dentistry studies and some dentistry areas that have never been explored by proteomic tools. It is expected that a greater integration of these areas will help to understand what is still unknown in oral health and disease. Copyright © 2013 Wiley Periodicals, Inc.

Life Stage-specific Proteomes of Legionella pneumophila Reveal a Highly Differential Abundance of Virulence-associated Dot/Icm effectors*

Science.gov (United States)

Aurass, Philipp; Gerlach, Thomas; Becher, Dörte; Voigt, Birgit; Karste, Susanne; Bernhardt, Jörg; Riedel, Katharina; Hecker, Michael; Flieger, Antje

2016-01-01

Major differences in the transcriptional program underlying the phenotypic switch between exponential and post-exponential growth of Legionella pneumophila were formerly described characterizing important alterations in infection capacity. Additionally, a third state is known where the bacteria transform in a viable but nonculturable state under stress, such as starvation. We here describe phase-related proteomic changes in exponential phase (E), postexponential phase (PE) bacteria, and unculturable microcosms (UNC) containing viable but nonculturable state cells, and identify phase-specific proteins. We present data on different bacterial subproteomes of E and PE, such as soluble whole cell proteins, outer membrane-associated proteins, and extracellular proteins. In total, 1368 different proteins were identified, 922 were quantified and 397 showed differential abundance in E/PE. The quantified subproteomes of soluble whole cell proteins, outer membrane-associated proteins, and extracellular proteins; 841, 55, and 77 proteins, respectively, were visualized in Voronoi treemaps. 95 proteins were quantified exclusively in E, such as cell division proteins MreC, FtsN, FtsA, and ZipA; 33 exclusively in PE, such as motility-related proteins of flagellum biogenesis FlgE, FlgK, and FliA; and 9 exclusively in unculturable microcosms soluble whole cell proteins, such as hypothetical, as well as transport/binding-, and metabolism-related proteins. A high frequency of differentially abundant or phase-exclusive proteins was observed among the 91 quantified effectors of the major virulence-associated protein secretion system Dot/Icm (> 60%). 24 were E-exclusive, such as LepA/B, YlfA, MavG, Lpg2271, and 13 were PE-exclusive, such as RalF, VipD, Lem10. The growth phase-related specific abundance of a subset of Dot/Icm virulence effectors was confirmed by means of Western blotting. We therefore conclude that many effectors are predominantly abundant at either E or PE which suggests

Biomarker Discovery and Mechanistic Studies of Prostate Cancer Using Targeted Proteomic Approaches

Science.gov (United States)

2010-07-01

EMMPRIN is implicated in metastasis via its ability to confer resistance of breast cancer cells to anoikis by inhibiting BIM [21], and its association with...of Bim . J Biol Chem 2006;281:9719–9727. 22. Gupta N, Wollscheid B, Watts JD, Scheer B, Aebersold R, DeFranco AL. Quantitative proteomic analysis of B...disseminated in electronic form, nor  deployed in part or in whole in any  marketing , promotional or educational  contexts without authorization from

Bioinformatics analysis of differentially expressed proteins in prostate cancer based on proteomics data

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Chen C

2016-03-01

Full Text Available Chen Chen,1 Li-Guo Zhang,1 Jian Liu,1 Hui Han,1 Ning Chen,1 An-Liang Yao,1 Shao-San Kang,1 Wei-Xing Gao,1 Hong Shen,2 Long-Jun Zhang,1 Ya-Peng Li,1 Feng-Hong Cao,1 Zhi-Guo Li3 1Department of Urology, North China University of Science and Technology Affiliated Hospital, 2Department of Modern Technology and Education Center, 3Department of Medical Research Center, International Science and Technology Cooperation Base of Geriatric Medicine, North China University of Science and Technology, Tangshan, People’s Republic of China Abstract: We mined the literature for proteomics data to examine the occurrence and metastasis of prostate cancer (PCa through a bioinformatics analysis. We divided the differentially expressed proteins (DEPs into two groups: the group consisting of PCa and benign tissues (P&b and the group presenting both high and low PCa metastatic tendencies (H&L. In the P&b group, we found 320 DEPs, 20 of which were reported more than three times, and DES was the most commonly reported. Among these DEPs, the expression levels of FGG, GSN, SERPINC1, TPM1, and TUBB4B have not yet been correlated with PCa. In the H&L group, we identified 353 DEPs, 13 of which were reported more than three times. Among these DEPs, MDH2 and MYH9 have not yet been correlated with PCa metastasis. We further confirmed that DES was differentially expressed between 30 cancer and 30 benign tissues. In addition, DEPs associated with protein transport, regulation of actin cytoskeleton, and the extracellular matrix (ECM–receptor interaction pathway were prevalent in the H&L group and have not yet been studied in detail in this context. Proteins related to homeostasis, the wound-healing response, focal adhesions, and the complement and coagulation pathways were overrepresented in both groups. Our findings suggest that the repeatedly reported DEPs in the two groups may function as potential biomarkers for detecting PCa and predicting its aggressiveness. Furthermore

Global Proteome Analysis of Leptospira interrogans

Science.gov (United States)

Comparative global proteome analyses were performed on Leptospira interrogans serovar Copenhageni grown under conventional in vitro conditions and those mimicking in vivo conditions (iron limitation and serum presence). Proteomic analyses were conducted using iTRAQ and LC-ESI-tandem mass spectrometr...

Comprehensive proteome analysis of lysosomes reveals the diverse function of macrophages in immune responses.

Science.gov (United States)

Gao, Yanpan; Chen, Yanyu; Zhan, Shaohua; Zhang, Wenhao; Xiong, Feng; Ge, Wei

2017-01-31

Phagocytosis and autophagy in macrophages have been shown to be essential to both innate and adaptive immunity. Lysosomes are the main catabolic subcellular organelles responsible for degradation and recycling of both extracellular and intracellular material, which are the final steps in phagocytosis and autophagy. However, the molecular mechanisms underlying lysosomal functions after infection remain obscure. In this study, we conducted a quantitative proteomics analysis of the changes in constitution and glycosylation of proteins in lysosomes derived from murine RAW 264.7 macrophage cells treated with different types of pathogens comprising examples of bacteria (Listeria monocytogenes, L. m), DNA viruses (herpes simplex virus type-1, HSV-1) and RNA viruses (vesicular stomatitis virus, VSV). In total, 3,704 lysosome-related proteins and 300 potential glycosylation sites on 193 proteins were identified. Comparative analysis showed that the aforementioned pathogens induced distinct alterations in the proteome of the lysosome, which is closely associated with the immune functions of macrophages, such as toll-like receptor activation, inflammation and antigen-presentation. The most significant changes in proteins and fluctuations in glycosylation were also determined. Furthermore, Western blot analysis showed that the changes in expression of these proteins were undetectable at the whole cell level. Thus, our study provides unique insights into the function of lysosomes in macrophage activation and immune responses.

Muscle Tissue Damage Induced by the Venom of Bothrops asper: Identification of Early and Late Pathological Events through Proteomic Analysis.

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Cristina Herrera

2016-04-01

Full Text Available The time-course of the pathological effects induced by the venom of the snake Bothrops asper in muscle tissue was investigated by a combination of histology, proteomic analysis of exudates collected in the vicinity of damaged muscle, and immunodetection of extracellular matrix proteins in exudates. Proteomic assay of exudates has become an excellent new methodological tool to detect key biomarkers of tissue alterations for a more integrative perspective of snake venom-induced pathology. The time-course analysis of the intracellular proteins showed an early presence of cytosolic and mitochondrial proteins in exudates, while cytoskeletal proteins increased later on. This underscores the rapid cytotoxic effect of venom, especially in muscle fibers, due to the action of myotoxic phospholipases A2, followed by the action of proteinases in the cytoskeleton of damaged muscle fibers. Similarly, the early presence of basement membrane (BM and other extracellular matrix (ECM proteins in exudates reflects the rapid microvascular damage and hemorrhage induced by snake venom metalloproteinases. The presence of fragments of type IV collagen and perlecan one hour after envenoming suggests that hydrolysis of these mechanically/structurally-relevant BM components plays a key role in the genesis of hemorrhage. On the other hand, the increment of some ECM proteins in the exudate at later time intervals is likely a consequence of the action of endogenous matrix metalloproteinases (MMPs or of de novo synthesis of ECM proteins during tissue remodeling as part of the inflammatory reaction. Our results offer relevant insights for a more integrative and systematic understanding of the time-course dynamics of muscle tissue damage induced by B. asper venom and possibly other viperid venoms.

Marine proteomics: a critical assessment of an emerging technology.

Science.gov (United States)

Slattery, Marc; Ankisetty, Sridevi; Corrales, Jone; Marsh-Hunkin, K Erica; Gochfeld, Deborah J; Willett, Kristine L; Rimoldi, John M

2012-10-26

The application of proteomics to marine sciences has increased in recent years because the proteome represents the interface between genotypic and phenotypic variability and, thus, corresponds to the broadest possible biomarker for eco-physiological responses and adaptations. Likewise, proteomics can provide important functional information regarding biosynthetic pathways, as well as insights into mechanism of action, of novel marine natural products. The goal of this review is to (1) explore the application of proteomics methodologies to marine systems, (2) assess the technical approaches that have been used, and (3) evaluate the pros and cons of this proteomic research, with the intent of providing a critical analysis of its future roles in marine sciences. To date, proteomics techniques have been utilized to investigate marine microbe, plant, invertebrate, and vertebrate physiology, developmental biology, seafood safety, susceptibility to disease, and responses to environmental change. However, marine proteomics studies often suffer from poor experimental design, sample processing/optimization difficulties, and data analysis/interpretation issues. Moreover, a major limitation is the lack of available annotated genomes and proteomes for most marine organisms, including several "model species". Even with these challenges in mind, there is no doubt that marine proteomics is a rapidly expanding and powerful integrative molecular research tool from which our knowledge of the marine environment, and the natural products from this resource, will be significantly expanded.

Proteogenomics Dashboard for the Human Proteome Project.

Science.gov (United States)

Tabas-Madrid, Daniel; Alves-Cruzeiro, Joao; Segura, Victor; Guruceaga, Elizabeth; Vialas, Vital; Prieto, Gorka; García, Carlos; Corrales, Fernando J; Albar, Juan Pablo; Pascual-Montano, Alberto

2015-09-04

dasHPPboard is a novel proteomics-based dashboard that collects and reports the experiments produced by the Spanish Human Proteome Project consortium (SpHPP) and aims to help HPP to map the entire human proteome. We have followed the strategy of analog genomics projects like the Encyclopedia of DNA Elements (ENCODE), which provides a vast amount of data on human cell lines experiments. The dashboard includes results of shotgun and selected reaction monitoring proteomics experiments, post-translational modifications information, as well as proteogenomics studies. We have also processed the transcriptomics data from the ENCODE and Human Body Map (HBM) projects for the identification of specific gene expression patterns in different cell lines and tissues, taking special interest in those genes having little proteomic evidence available (missing proteins). Peptide databases have been built using single nucleotide variants and novel junctions derived from RNA-Seq data that can be used in search engines for sample-specific protein identifications on the same cell lines or tissues. The dasHPPboard has been designed as a tool that can be used to share and visualize a combination of proteomic and transcriptomic data, providing at the same time easy access to resources for proteogenomics analyses. The dasHPPboard can be freely accessed at: http://sphppdashboard.cnb.csic.es.

The International Society for Extracellular Vesicles launches the first massive open online course on extracellular vesicles

OpenAIRE

L?sser, Cecilia; Th?ry, Clotilde; Buz?s, Edit I.; Mathivanan, Suresh; Zhao, Weian; Gho, Yong Song; L?tvall, Jan

2016-01-01

The International Society for Extracellular Vesicles (ISEV) has organised its first educational online course for students and beginners in the field of extracellular vesicles (EVs). This course, “Basics of Extracellular Vesicles,” uses recorded lectures from experts in the field and will be open for an unlimited number of participants. The course is divided into 5 modules and can be accessed at www.coursera.org/learn/extracellular-vesicles. The first module is an introduction to the field co...

Proteomic Biomarkers for Spontaneous Preterm Birth

DEFF Research Database (Denmark)

Kacerovsky, Marian; Lenco, Juraj; Musilova, Ivana

2014-01-01

This review aimed to identify, synthesize, and analyze the findings of studies on proteomic biomarkers for spontaneous preterm birth (PTB). Three electronic databases (Medline, Embase, and Scopus) were searched for studies in any language reporting the use of proteomic biomarkers for PTB published...

Extracellular tumor-related mRNA in plasma of lymphoma patients and survival implications.

Directory of Open Access Journals (Sweden)

Vanesa Garcia

Full Text Available BACKGROUND: We studied anomalous extracellular mRNAs in plasma from patients with diffuse large B-cell lymphoma (DLBCL and their survival implications. mRNAs studied have been reported in the literature as markers of poor (BCL2, CCND2, MYC and favorable outcome (LMO2, BCL6, FN1 in tumors. These markers were also analyzed in lymphoma tissues to test possible associations with their presence in plasma. METHODOLOGY/PRINCIPAL FINDINGS: mRNA from 42 plasma samples and 12 tumors from patients with DLBCL was analyzed by real-time PCR. Samples post-treatment were studied. The immunohistochemistry of BCL2 and BCL6 was defined. Presence of circulating tumor cells was determined by analyzing the clonality of the immunoglobulin heavy-chain genes by PCR. In DLBCL, MYC mRNA was associated with short overall survival. mRNA targets with unfavorable outcome in tumors were associated with characteristics indicative of poor prognosis, with partial treatment response and with short progression-free survival in patients with complete response. In patients with low IPI score, unfavorable mRNA targets were related to shorter overall survival, partial response, high LDH levels and death. mRNA disappeared in post-treatment samples of patients with complete response, and persisted in those with partial response or death. No associations were found between circulating tumor cells and plasma mRNA. Absence of BCL6 protein in tumors was associated with presence of unfavorable plasma mRNA. CONCLUSIONS/SIGNIFICANCE: Through a non-invasive procedure, tumor-derived mRNAs can be obtained in plasma. mRNA detected in plasma did not proceed from circulating tumor cells. In our study, unfavorable targets in plasma were associated with poor prognosis in B-cell lymphomas, mainly MYC mRNA. Moreover, the unfavorable targets in plasma could help us to classify patients with poor outcome within the good prognosis group according to IPI.

Cellular therapy without cells: extracellular vesicles promote activation of stem cells after irradiation

International Nuclear Information System (INIS)

Lange, C.

2016-01-01

Mesenchymal stromal cells from the bone marrow (MSC) have been shown to be effective in several cell therapeutic treatments. However, MSC accumulate in lungs after i.v. injection. How do MSC transfer their potential to organs with therapeutic need? We show that released extracellular vesicles (EV) might be playing an active role in this transfer. EV were isolated from MSC supernatant and characterized with flow cytometry, proteomics and next generation sequencing. Our data showed the transfer of RNAs, clustering into several protective gene groups. Besides, we repeatedly detected genomic DNA on vesicles. Using a plant - derived detector gene we showed horizontal DNA transfer via EV. Furthermore, we showed that EV were able to salvage stem/progenitor cells in vitro from radiation suppression. Three selected proteins from proteomics data were examined for stem cell protection after irradiation. EV derived from down-regulated producer MSC showed a substantial loss of protection in irradiated stem cells supporting their relevance for stem cell protection. Finally, we showed that EV after i.v. injection into lethally irradiated animals colocalize within 2-4 hours with hematopoietic stem cells in the bone marrow giving hint to direct protection of stem cells by EV. In conclusion, EV derived from bone marrow MSC were able to transfer several cargo compounds leading potentially to change of the genetic properties. Importantly, EV protect irradiated hematopoietic stem cells, stimulate their recovery and proliferation and rescue lethally irradiated animals long-term. Thus, EV might be an alternative for future cell therapeutic treatment particularly in radiation-based events. (author)

Urine proteome analysis in Dent's disease shows high selective changes potentially involved in chronic renal damage.

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Santucci, Laura; Candiano, Giovanni; Anglani, Franca; Bruschi, Maurizio; Tosetto, Enrica; Cremasco, Daniela; Murer, Luisa; D'Ambrosio, Chiara; Scaloni, Andrea; Petretto, Andrea; Caridi, Gianluca; Rossi, Roberta; Bonanni, Alice; Ghiggeri, Gian Marco

2016-01-01

Definition of the urinary protein composition would represent a potential tool for diagnosis in many clinical conditions. The use of new proteomic technologies allows detection of genetic and post-trasductional variants that increase sensitivity of the approach but complicates comparison within a heterogeneous patient population. Overall, this limits research of urinary biomarkers. Studying monogenic diseases are useful models to address this issue since genetic variability is reduced among first- and second-degree relatives of the same family. We applied this concept to Dent's disease, a monogenic condition characterised by low-molecular-weight proteinuria that is inherited following an X-linked trait. Results are presented here on a combined proteomic approach (LC-mass spectrometry, Western blot and zymograms for proteases and inhibitors) to characterise urine proteins in a large family (18 members, 6 hemizygous patients, 6 carrier females, and 6 normals) with Dent's diseases due to the 1070G>T mutation of the CLCN5. Gene ontology analysis on more than 1000 proteins showed that several clusters of proteins characterised urine of affected patients compared to carrier females and normal subjects: proteins involved in extracellular matrix remodelling were the major group. Specific analysis on metalloproteases and their inhibitors underscored unexpected mechanisms potentially involved in renal fibrosis. Studying with new-generation techniques for proteomic analysis of the members of a large family with Dent's disease sharing the same molecular defect allowed highly repetitive results that justify conclusions. Identification in urine of proteins actively involved in interstitial matrix remodelling poses the question of active anti-fibrotic drugs in Dent's patients. Copyright © 2015 Elsevier B.V. All rights reserved.

Proteomic overview and perspectives of the radiation-induced bystander effects.

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Chevalier, François; Hamdi, Dounia Houria; Saintigny, Yannick; Lefaix, Jean-Louis

2015-01-01

Radiation proteomics is a recent, promising and powerful tool to identify protein markers of direct and indirect consequences of ionizing radiation. The main challenges of modern radiobiology is to predict radio-sensitivity of patients and radio-resistance of tumor to be treated, but considerable evidences are now available regarding the significance of a bystander effect at low and high doses. This "radiation-induced bystander effect" (RIBE) is defined as the biological responses of non-irradiated cells that received signals from neighboring irradiated cells. Such intercellular signal is no more considered as a minor side-effect of radiotherapy in surrounding healthy tissue and its occurrence should be considered in adapting radiotherapy protocols, to limit the risk for radiation-induced secondary cancer. There is no consensus on a precise designation of RIBE, which involves a number of distinct signal-mediated effects within or outside the irradiated volume. Indeed, several cellular mechanisms were proposed, including the secretion of soluble factors by irradiated cells in the extracellular matrix, or the direct communication between irradiated and neighboring non-irradiated cells via gap junctions. This phenomenon is observed in a context of major local inflammation, linked with a global imbalance of oxidative metabolism which makes its analysis challenging using in vitro model systems. In this review article, the authors first define the radiation-induced bystander effect as a function of radiation type, in vitro analysis protocols, and cell type. In a second time, the authors present the current status of protein biomarkers and proteomic-based findings and discuss the capacities, limits and perspectives of such global approaches to explore these complex intercellular mechanisms. Copyright © 2014 Elsevier B.V. All rights reserved.

Kalirin and CHD7: novel endothelial dysfunction indicators in circulating extracellular vesicles from hypertensive patients with albuminuria

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de la Cuesta, Fernando; Baldan-Martin, Montserrat; Moreno-Luna, Rafael; Alvarez-Llamas, Gloria; Gonzalez-Calero, Laura; Mourino-Alvarez, Laura; Sastre-Oliva, Tamara; López, Juan A.; Vázquez, Jesús; Ruiz-Hurtado, Gema; Segura, Julian; Vivanco, Fernando; Ruilope, Luis M.; Barderas, Maria G.

2017-01-01

Despite of the great advances in anti-hypertensive therapies, many patients under Renin-Angiotensin- System (RAS) suppression develop albuminuria, which is a clear indicator of therapeutic inefficiency. Hence, indicators of vascular function are needed to assess patients’ condition and help deciding future therapies. Proteomic analysis of circulating extracellular vesicles (EVs) showed two proteins, kalirin and chromodomain-helicase-DNA-binding protein 7 (CHD7), increased in albuminuric patients. A positive correlation of both with the expression of the endothelial activation marker E-selectin was found in EVs. In vitro analysis using TNFα-treated adult human endothelial cells proved their involvement in endothelial cell activation. Hence, we propose protein levels of kalirin and CHD7 in circulating EVs as novel endothelial dysfunction markers to monitor vascular condition in hypertensive patients with albuminuria. PMID:28152519

PCAS – a precomputed proteome annotation database resource

Directory of Open Access Journals (Sweden)

Luo Jingchu

2003-11-01

Full Text Available Abstract Background Many model proteomes or "complete" sets of proteins of given organisms are now publicly available. Much effort has been invested in computational annotation of those "draft" proteomes. Motif or domain based algorithms play a pivotal role in functional classification of proteins. Employing most available computational algorithms, mainly motif or domain recognition algorithms, we set up to develop an online proteome annotation system with integrated proteome annotation data to complement existing resources. Results We report here the development of PCAS (ProteinCentric Annotation System as an online resource of pre-computed proteome annotation data. We applied most available motif or domain databases and their analysis methods, including hmmpfam search of HMMs in Pfam, SMART and TIGRFAM, RPS-PSIBLAST search of PSSMs in CDD, pfscan of PROSITE patterns and profiles, as well as PSI-BLAST search of SUPERFAMILY PSSMs. In addition, signal peptide and TM are predicted using SignalP and TMHMM respectively. We mapped SUPERFAMILY and COGs to InterPro, so the motif or domain databases are integrated through InterPro. PCAS displays table summaries of pre-computed data and a graphical presentation of motifs or domains relative to the protein. As of now, PCAS contains human IPI, mouse IPI, and rat IPI, A. thaliana, C. elegans, D. melanogaster, S. cerevisiae, and S. pombe proteome. PCAS is available at http://pak.cbi.pku.edu.cn/proteome/gca.php Conclusion PCAS gives better annotation coverage for model proteomes by employing a wider collection of available algorithms. Besides presenting the most confident annotation data, PCAS also allows customized query so users can inspect statistically less significant boundary information as well. Therefore, besides providing general annotation information, PCAS could be used as a discovery platform. We plan to update PCAS twice a year. We will upgrade PCAS when new proteome annotation algorithms

Improving HIV proteome annotation: new features of BioAfrica HIV Proteomics Resource.

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Druce, Megan; Hulo, Chantal; Masson, Patrick; Sommer, Paula; Xenarios, Ioannis; Le Mercier, Philippe; De Oliveira, Tulio

2016-01-01

The Human Immunodeficiency Virus (HIV) is one of the pathogens that cause the greatest global concern, with approximately 35 million people currently infected with HIV. Extensive HIV research has been performed, generating a large amount of HIV and host genomic data. However, no effective vaccine that protects the host from HIV infection is available and HIV is still spreading at an alarming rate, despite effective antiretroviral (ARV) treatment. In order to develop effective therapies, we need to expand our knowledge of the interaction between HIV and host proteins. In contrast to virus proteins, which often rapidly evolve drug resistance mutations, the host proteins are essentially invariant within all humans. Thus, if we can identify the host proteins needed for virus replication, such as those involved in transporting viral proteins to the cell surface, we have a chance of interrupting viral replication. There is no proteome resource that summarizes this interaction, making research on this subject a difficult enterprise. In order to fill this gap in knowledge, we curated a resource presents detailed annotation on the interaction between the HIV proteome and host proteins. Our resource was produced in collaboration with ViralZone and used manual curation techniques developed by UniProtKB/Swiss-Prot. Our new website also used previous annotations of the BioAfrica HIV-1 Proteome Resource, which has been accessed by approximately 10 000 unique users a year since its inception in 2005. The novel features include a dedicated new page for each HIV protein, a graphic display of its function and a section on its interaction with host proteins. Our new webpages also add information on the genomic location of each HIV protein and the position of ARV drug resistance mutations. Our improved BioAfrica HIV-1 Proteome Resource fills a gap in the current knowledge of biocuration.Database URL:http://www.bioafrica.net/proteomics/HIVproteome.html. © The Author(s) 2016. Published

Proteomics and the dynamic plasma membrane

DEFF Research Database (Denmark)

Sprenger, Richard R; Jensen, Ole Nørregaard

2010-01-01

plasma membrane is of particular interest, by not only serving as a barrier between the "cell interior" and the external environment, but moreover by organizing and clustering essential components to enable dynamic responses to internal and external stimuli. Defining and characterizing the dynamic plasma...... the challenges in functional proteomic studies of the plasma membrane. We review the recent progress in MS-based plasma membrane proteomics by presenting key examples from eukaryotic systems, including mammals, yeast and plants. We highlight the importance of enrichment and quantification technologies required...... for detailed functional and comparative analysis of the dynamic plasma membrane proteome....

PROTEOMICS in aquaculture: applications and trends.

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Rodrigues, Pedro M; Silva, Tomé S; Dias, Jorge; Jessen, Flemming

2012-07-19

Over the last forty years global aquaculture presented a growth rate of 6.9% per annum with an amazing production of 52.5 million tonnes in 2008, and a contribution of 43% of aquatic animal food for human consumption. In order to meet the world's health requirements of fish protein, a continuous growth in production is still expected for decades to come. Aquaculture is, though, a very competitive market, and a global awareness regarding the use of scientific knowledge and emerging technologies to obtain a better farmed organism through a sustainable production has enhanced the importance of proteomics in seafood biology research. Proteomics, as a powerful comparative tool, has therefore been increasingly used over the last decade to address different questions in aquaculture, regarding welfare, nutrition, health, quality, and safety. In this paper we will give an overview of these biological questions and the role of proteomics in their investigation, outlining the advantages, disadvantages and future challenges. A brief description of the proteomics technical approaches will be presented. Special focus will be on the latest trends related to the aquaculture production of fish with defined nutritional, health or quality properties for functional foods and the integration of proteomics techniques in addressing this challenging issue. Copyright © 2012 Elsevier B.V. All rights reserved.

Proteomics in uveal melanoma.

LENUS (Irish Health Repository)

Ramasamy, Pathma

2014-01-01

Uveal melanoma is the most common primary intraocular malignancy in adults, with an incidence of 5-7 per million per year. It is associated with the development of metastasis in about 50% of cases, and 40% of patients with uveal melanoma die of metastatic disease despite successful treatment of the primary tumour. The survival rates at 5, 10 and 15 years are 65%, 50% and 45% respectively. Unlike progress made in many other areas of cancer, uveal melanoma is still poorly understood and survival rates have remained similar over the past 25 years. Recently, advances made in molecular genetics have improved our understanding of this disease and stratification of patients into low risk and high risk for developing metastasis. However, only a limited number of studies have been performed using proteomic methods. This review will give an overview of various proteomic technologies currently employed in life sciences research, and discuss proteomic studies of uveal melanoma.

CPTAC Collaborates with Molecular & Cellular Proteomics to Address Reproducibility in Targeted Assay Development | Office of Cancer Clinical Proteomics Research

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The journal Molecular & Cellular Proteomics (MCP), in collaboration with the Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI), part of the National Institutes of Health, announce new guidelines and requirements for papers describing the development and application of targeted mass spectrometry measurements of peptides, modified peptides and proteins (Mol Cell Proteomics 2017; PMID: 28183812).  NCI’s participation is part of NIH’s overall effort to address the r

Challenges for proteomics core facilities.

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Lilley, Kathryn S; Deery, Michael J; Gatto, Laurent

2011-03-01

Many analytical techniques have been executed by core facilities established within academic, pharmaceutical and other industrial institutions. The centralization of such facilities ensures a level of expertise and hardware which often cannot be supported by individual laboratories. The establishment of a core facility thus makes the technology available for multiple researchers in the same institution. Often, the services within the core facility are also opened out to researchers from other institutions, frequently with a fee being levied for the service provided. In the 1990s, with the onset of the age of genomics, there was an abundance of DNA analysis facilities, many of which have since disappeared from institutions and are now available through commercial sources. Ten years on, as proteomics was beginning to be utilized by many researchers, this technology found itself an ideal candidate for being placed within a core facility. We discuss what in our view are the daily challenges of proteomics core facilities. We also examine the potential unmet needs of the proteomics core facility that may also be applicable to proteomics laboratories which do not function as core facilities. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitative proteomics and systems analysis of cultured H9C2 cardiomyoblasts during differentiation over time supports a 'function follows form' model of differentiation.

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Kankeu, Cynthia; Clarke, Kylie; Van Haver, Delphi; Gevaert, Kris; Impens, Francis; Dittrich, Anna; Roderick, H Llewelyn; Passante, Egle; Huber, Heinrich J

2018-05-17

The rat cardiomyoblast cell line H9C2 has emerged as a valuable tool for studying cardiac development, mechanisms of disease and toxicology. We present here a rigorous proteomic analysis that monitored the changes in protein expression during differentiation of H9C2 cells into cardiomyocyte-like cells over time. Quantitative mass spectrometry followed by gene ontology (GO) enrichment analysis revealed that early changes in H9C2 differentiation are related to protein pathways of cardiac muscle morphogenesis and sphingolipid synthesis. These changes in the proteome were followed later in the differentiation time-course by alterations in the expression of proteins involved in cation transport and beta-oxidation. Studying the temporal profile of the H9C2 proteome during differentiation in further detail revealed eight clusters of co-regulated proteins that can be associated with early, late, continuous and transient up- and downregulation. Subsequent reactome pathway analysis based on these eight clusters further corroborated and detailed the results of the GO analysis. Specifically, this analysis confirmed that proteins related to pathways in muscle contraction are upregulated early and transiently, and proteins relevant to extracellular matrix organization are downregulated early. In contrast, upregulation of proteins related to cardiac metabolism occurs at later time points. Finally, independent validation of the proteomics results by immunoblotting confirmed hereto unknown regulators of cardiac structure and ionic metabolism. Our results are consistent with a 'function follows form' model of differentiation, whereby early and transient alterations of structural proteins enable subsequent changes that are relevant to the characteristic physiology of cardiomyocytes.

Proteomic approach toward molecular backgrounds of drug resistance of osteosarcoma cells in spheroid culture system.

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Arai, Kazuya; Sakamoto, Ruriko; Kubota, Daisuke; Kondo, Tadashi

2013-08-01

Chemoresistance is one of the most critical prognostic factors in osteosarcoma, and elucidation of the molecular backgrounds of chemoresistance may lead to better clinical outcomes. Spheroid cells resemble in vivo cells and are considered an in vitro model for the drug discovery. We found that spheroid cells displayed more chemoresistance than conventional monolayer cells across 11 osteosarcoma cell lines. To investigate the molecular mechanisms underlying the resistance to chemotherapy, we examined the proteomic differences between the monolayer and spheroid cells by 2D-DIGE. Of the 4762 protein species observed, we further investigated 435 species with annotated mass spectra in the public proteome database, Genome Medicine Database of Japan Proteomics. Among the 435 protein species, we found that 17 species exhibited expression level differences when the cells formed spheroids in more than five cell lines and four species out of these 17 were associated with spheroid-formation associated resistance to doxorubicin. We confirmed the upregulation of cathepsin D in spheroid cells by western blotting. Cathepsin D has been implicated in chemoresistance of various malignancies but has not previously been implemented in osteosarcoma. Our study suggested that the spheroid system may be a useful tool to reveal the molecular backgrounds of chemoresistance in osteosarcoma. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteomic analysis of secreted proteins by human bronchial epithelial cells in response to cadmium toxicity.

Science.gov (United States)

Chen, De-Ju; Xu, Yan-Ming; Zheng, Wei; Huang, Dong-Yang; Wong, Wing-Yan; Tai, William Chi-Shing; Cho, Yong-Yeon; Lau, Andy T Y

2015-09-01

For years, many studies have been conducted to investigate the intracellular response of cells challenged with toxic metal(s), yet, the corresponding secretome responses, especially in human lung cells, are largely unexplored. Here, we provide a secretome analysis of human bronchial epithelial cells (BEAS-2B) treated with cadmium chloride (CdCl2 ), with the aim of identifying secreted proteins in response to Cd toxicity. Proteins from control and spent media were separated by two-dimensional electrophoresis and visualized by silver staining. Differentially-secreted proteins were identified by MALDI-TOF-MS analysis and database searching. We characterized, for the first time, the extracellular proteome changes of BEAS-2B dosed with Cd. Our results unveiled that Cd treatment led to the marked upregulation of molecular chaperones, antioxidant enzymes, enzymes associated with glutathione metabolic process, proteins involved in cellular energy metabolism, as well as tumor-suppressors. Pretreatment of cells with the thiol antioxidant glutathione before Cd treatment effectively abrogated the secretion of these proteins and prevented cell death. Taken together, our results demonstrate that Cd causes oxidative stress-induced cytotoxicity; and the differentially-secreted protein signatures could be considered as targets for potential use as extracellular biomarkers upon Cd exposure. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An individual urinary proteome analysis in normal human beings to define the minimal sample number to represent the normal urinary proteome

Directory of Open Access Journals (Sweden)

Liu Xuejiao

2012-11-01

Full Text Available Abstract Background The urinary proteome has been widely used for biomarker discovery. A urinary proteome database from normal humans can provide a background for discovery proteomics and candidate proteins/peptides for targeted proteomics. Therefore, it is necessary to define the minimum number of individuals required for sampling to represent the normal urinary proteome. Methods In this study, inter-individual and inter-gender variations of urinary proteome were taken into consideration to achieve a representative database. An individual analysis was performed on overnight urine samples from 20 normal volunteers (10 males and 10 females by 1DLC/MS/MS. To obtain a representative result of each sample, a replicate 1DLCMS/MS analysis was performed. The minimal sample number was estimated by statistical analysis. Results For qualitative analysis, less than 5% of new proteins/peptides were identified in a male/female normal group by adding a new sample when the sample number exceeded nine. In addition, in a normal group, the percentage of newly identified proteins/peptides was less than 5% upon adding a new sample when the sample number reached 10. Furthermore, a statistical analysis indicated that urinary proteomes from normal males and females showed different patterns. For quantitative analysis, the variation of protein abundance was defined by spectrum count and western blotting methods. And then the minimal sample number for quantitative proteomic analysis was identified. Conclusions For qualitative analysis, when considering the inter-individual and inter-gender variations, the minimum sample number is 10 and requires a balanced number of males and females in order to obtain a representative normal human urinary proteome. For quantitative analysis, the minimal sample number is much greater than that for qualitative analysis and depends on the experimental methods used for quantification.

Proteomic approaches in research of cyanobacterial photosynthesis.

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Battchikova, Natalia; Angeleri, Martina; Aro, Eva-Mari

2015-10-01

Oxygenic photosynthesis in cyanobacteria, algae, and plants is carried out by a fabulous pigment-protein machinery that is amazingly complicated in structure and function. Many different approaches have been undertaken to characterize the most important aspects of photosynthesis, and proteomics has become the essential component in this research. Here we describe various methods which have been used in proteomic research of cyanobacteria, and demonstrate how proteomics is implemented into on-going studies of photosynthesis in cyanobacterial cells.

Proteomics wants cRacker: automated standardized data analysis of LC-MS derived proteomic data.

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Zauber, Henrik; Schulze, Waltraud X

2012-11-02

The large-scale analysis of thousands of proteins under various experimental conditions or in mutant lines has gained more and more importance in hypothesis-driven scientific research and systems biology in the past years. Quantitative analysis by large scale proteomics using modern mass spectrometry usually results in long lists of peptide ion intensities. The main interest for most researchers, however, is to draw conclusions on the protein level. Postprocessing and combining peptide intensities of a proteomic data set requires expert knowledge, and the often repetitive and standardized manual calculations can be time-consuming. The analysis of complex samples can result in very large data sets (lists with several 1000s to 100,000 entries of different peptides) that cannot easily be analyzed using standard spreadsheet programs. To improve speed and consistency of the data analysis of LC-MS derived proteomic data, we developed cRacker. cRacker is an R-based program for automated downstream proteomic data analysis including data normalization strategies for metabolic labeling and label free quantitation. In addition, cRacker includes basic statistical analysis, such as clustering of data, or ANOVA and t tests for comparison between treatments. Results are presented in editable graphic formats and in list files.

Automation, parallelism, and robotics for proteomics.

Science.gov (United States)

Alterovitz, Gil; Liu, Jonathan; Chow, Jijun; Ramoni, Marco F

2006-07-01

The speed of the human genome project (Lander, E. S., Linton, L. M., Birren, B., Nusbaum, C. et al., Nature 2001, 409, 860-921) was made possible, in part, by developments in automation of sequencing technologies. Before these technologies, sequencing was a laborious, expensive, and personnel-intensive task. Similarly, automation and robotics are changing the field of proteomics today. Proteomics is defined as the effort to understand and characterize proteins in the categories of structure, function and interaction (Englbrecht, C. C., Facius, A., Comb. Chem. High Throughput Screen. 2005, 8, 705-715). As such, this field nicely lends itself to automation technologies since these methods often require large economies of scale in order to achieve cost and time-saving benefits. This article describes some of the technologies and methods being applied in proteomics in order to facilitate automation within the field as well as in linking proteomics-based information with other related research areas.

Comparative Proteomic Profile of the Human Umbilical Cord Blood Exosomes between Normal and Preeclampsia Pregnancies with High-Resolution Mass Spectrometry

Directory of Open Access Journals (Sweden)

Ruizhe Jia

2015-07-01

Full Text Available Background/Aims: Exosomes are extracellular vesicles that are involved in several biological processes. The roles of proteins from human umbilical cord blood exosomes in the pathogenesis of preeclampsia remains poorly understood. Methods: In this study, we used high-resolution LC-MS/MS technologies to construct a comparative proteomic profiling of human umbilical cord blood exosomes between normal and preeclamptic pregnancies. Results: A total of 221 proteins were detected in human umbilical cord blood exosomes, with 14 upregulated and 15 downregulated proteins were definitively identified between preeclamptic and control pregnancies. Further bioinformatics analysis (Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that these differentially expressed proteins correlate with enzyme regulator activity, binding, extracellular region, cell part, biological regulation, cellular process and complement and coagulation cascades occurring during pathological changes of preeclampsia. Conclusion: Our results show significantly altered expression profiles of proteins in human umbilical cord blood exosomes between normal and preeclampsia pregnancies. These proteins may be involved in the etiology of preeclampsia.

Expanding the bovine milk proteome through extensive fractionation

DEFF Research Database (Denmark)

Nissen, Asger; Bendixen, Emøke; Ingvartsen, Klaus Lønne

2013-01-01

Bovine milk is an agricultural product of tremendous value worldwide. It contains proteins, fat, lactose, vitamins, and minerals. It provides nutrition and immunological protection (e.g., in the gastrointestinal tract) to the newborn and young calf. It also forms an important part of human...... of low abundant proteins. Further, the general health and udder health of the dairy cows delivering the milk may influence the composition of the milk proteome. To gain a more exhaustive and true picture of the milk proteome, we performed an extensive preanalysis fractionation of raw composite milk...... nutrition. The repertoire of proteins in milk (i.e., its proteome) is vast and complex. The milk proteome can be described in detail by mass spectrometry-based proteomics. However, the high concentration of dominating proteins in milk reduces mass spectrometry detection sensitivity and limits detection...

Extracellular Actin Is a Receptor for Mycoplasma hyopneumoniae

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Benjamin B. A. Raymond

2018-02-01

Full Text Available Mycoplasma hyopneumoniae, an agriculturally important porcine pathogen, disrupts the mucociliary escalator causing ciliostasis, loss of cilial function, and epithelial cell death within the porcine lung. Losses to swine production due to growth rate retardation and reduced feed conversion efficiency are severe, and antibiotics are used heavily to control mycoplasmal pneumonia. Notably, little is known about the repertoire of host receptors that M. hyopneumoniae targets to facilitate colonization. Here we show, for the first time, that actin exists extracellularly on porcine epithelial monolayers (PK-15 using surface biotinylation and 3D-Structured Illumination Microscopy (3D-SIM, and that M. hyopneumoniae binds to the extracellular β-actin exposed on the surface of these cells. Consistent with this hypothesis we show: (i monoclonal antibodies that target β-actin significantly block the ability of M. hyopneumoniae to adhere and colonize PK-15 cells; (ii microtiter plate binding assays show that M. hyopneumoniae cells bind to monomeric G-actin in a dose dependent manner; (iii more than 100 M. hyopneumoniae proteins were recovered from affinity-chromatography experiments using immobilized actin as bait; and (iv biotinylated monomeric actin binds directly to M. hyopneumoniae proteins in ligand blotting studies. Specifically, we show that the P97 cilium adhesin possesses at least two distinct actin-binding regions, and binds monomeric actin with nanomolar affinity. Taken together, these observations suggest that actin may be an important receptor for M. hyopneumoniae within the swine lung and will aid in the future development of intervention strategies against this devastating pathogen. Furthermore, our observations have wider implications for extracellular actin as an important bacterial receptor.

Extracellular Actin Is a Receptor for Mycoplasma hyopneumoniae.

Science.gov (United States)

Raymond, Benjamin B A; Madhkoor, Ranya; Schleicher, Ina; Uphoff, Cord C; Turnbull, Lynne; Whitchurch, Cynthia B; Rohde, Manfred; Padula, Matthew P; Djordjevic, Steven P

2018-01-01

Mycoplasma hyopneumoniae , an agriculturally important porcine pathogen, disrupts the mucociliary escalator causing ciliostasis, loss of cilial function, and epithelial cell death within the porcine lung. Losses to swine production due to growth rate retardation and reduced feed conversion efficiency are severe, and antibiotics are used heavily to control mycoplasmal pneumonia. Notably, little is known about the repertoire of host receptors that M. hyopneumoniae targets to facilitate colonization. Here we show, for the first time, that actin exists extracellularly on porcine epithelial monolayers (PK-15) using surface biotinylation and 3D-Structured Illumination Microscopy (3D-SIM), and that M. hyopneumoniae binds to the extracellular β-actin exposed on the surface of these cells. Consistent with this hypothesis we show: (i) monoclonal antibodies that target β-actin significantly block the ability of M. hyopneumoniae to adhere and colonize PK-15 cells; (ii) microtiter plate binding assays show that M. hyopneumoniae cells bind to monomeric G-actin in a dose dependent manner; (iii) more than 100 M. hyopneumoniae proteins were recovered from affinity-chromatography experiments using immobilized actin as bait; and (iv) biotinylated monomeric actin binds directly to M. hyopneumoniae proteins in ligand blotting studies. Specifically, we show that the P97 cilium adhesin possesses at least two distinct actin-binding regions, and binds monomeric actin with nanomolar affinity. Taken together, these observations suggest that actin may be an important receptor for M. hyopneumoniae within the swine lung and will aid in the future development of intervention strategies against this devastating pathogen. Furthermore, our observations have wider implications for extracellular actin as an important bacterial receptor.

Spermatogenesis in mammals: proteomic insights.

Science.gov (United States)

Chocu, Sophie; Calvel, Pierre; Rolland, Antoine D; Pineau, Charles

2012-08-01

Spermatogenesis is a highly sophisticated process involved in the transmission of genetic heritage. It includes halving ploidy, repackaging of the chromatin for transport, and the equipment of developing spermatids and eventually spermatozoa with the advanced apparatus (e.g., tightly packed mitochondrial sheat in the mid piece, elongating of the tail, reduction of cytoplasmic volume) to elicit motility once they reach the epididymis. Mammalian spermatogenesis is divided into three phases. In the first the primitive germ cells or spermatogonia undergo a series of mitotic divisions. In the second the spermatocytes undergo two consecutive divisions in meiosis to produce haploid spermatids. In the third the spermatids differentiate into spermatozoa in a process called spermiogenesis. Paracrine, autocrine, juxtacrine, and endocrine pathways all contribute to the regulation of the process. The array of structural elements and chemical factors modulating somatic and germ cell activity is such that the network linking the various cellular activities during spermatogenesis is unimaginably complex. Over the past two decades, advances in genomics have greatly improved our knowledge of spermatogenesis, by identifying numerous genes essential for the development of functional male gametes. Large-scale analyses of testicular function have deepened our insight into normal and pathological spermatogenesis. Progress in genome sequencing and microarray technology have been exploited for genome-wide expression studies, leading to the identification of hundreds of genes differentially expressed within the testis. However, although proteomics has now come of age, the proteomics-based investigation of spermatogenesis remains in its infancy. Here, we review the state-of-the-art of large-scale proteomic analyses of spermatogenesis, from germ cell development during sex determination to spermatogenesis in the adult. Indeed, a few laboratories have undertaken differential protein profiling

Polyphemus, Odysseus and the ovine milk proteome.

Science.gov (United States)

Cunsolo, Vincenzo; Fasoli, Elisa; Di Francesco, Antonella; Saletti, Rosaria; Muccilli, Vera; Gallina, Serafina; Righetti, Pier Giorgio; Foti, Salvatore

2017-01-30

In the last years the amount of ovine milk production, mainly used to formulate a wide range of different and exclusive dairy products often categorized as gourmet food, has been progressively increasing. Taking also into account that sheep milk (SM) also appears to be potentially less allergenic than cow's one, an in-depth information about its protein composition is essential to improve the comprehension of its potential benefits for human consumption. The present work reports the results of an in-depth characterization of SM whey proteome, carried out by coupling the CPLL technology with SDS-PAGE and high resolution UPLC-nESI MS/MS analysis. This approach allowed the identification of 718 different protein components, 644 of which are from unique genes. Particularly, this identification has expanded literature data about sheep whey proteome by 193 novel proteins previously undetected, many of which are involved in the defence/immunity mechanisms or in the nutrient delivery system. A comparative analysis of SM proteome known to date with cow's milk proteome, evidenced that while about 29% of SM proteins are also present in CM, 71% of the identified components appear to be unique of SM proteome and include a heterogeneous group of components which seem to have health-promoting benefits. The data have been deposited to the ProteomeXchange with identifier . Copyright © 2016 Elsevier B.V. All rights reserved.

Proteomics analyses of human optic nerve head astrocytes following biomechanical strain.

Science.gov (United States)

Rogers, Ronan S; Dharsee, Moyez; Ackloo, Suzanne; Sivak, Jeremy M; Flanagan, John G

2012-02-01

We investigate the role of glial cell activation in the human optic nerve caused by raised intraocular pressure, and their potential role in the development of glaucomatous optic neuropathy. To do this we present a proteomics study of the response of cultured, optic nerve head astrocytes to biomechanical strain, the magnitude and mode of strain based on previously published quantitative models. In this case, astrocytes were subjected to 3 and 12% stretches for either 2 h or 24 h. Proteomic methods included nano-liquid chromatography, tandem mass spectrometry, and iTRAQ labeling. Using controls for both stretch and time, a six-plex iTRAQ liquid chromatography- tandem MS (LC/MS/MS) experiment yielded 573 proteins discovered at a 95% confidence limit. The pathways included transforming growth factor β1, tumor necrosis factor, caspase 3, and tumor protein p53, which have all been implicated in the activation of astrocytes and are believed to play a role in the development of glaucomatous optic neuropathy. Confirmation of the iTRAQ analysis was performed by Western blotting of various proteins of interest including ANXA 4, GOLGA2, and αB-Crystallin.

Proteomics Analyses of Human Optic Nerve Head Astrocytes Following Biomechanical Strain*

Science.gov (United States)

Rogers, Ronan S.; Dharsee, Moyez; Ackloo, Suzanne; Sivak, Jeremy M.; Flanagan, John G.

2012-01-01

We investigate the role of glial cell activation in the human optic nerve caused by raised intraocular pressure, and their potential role in the development of glaucomatous optic neuropathy. To do this we present a proteomics study of the response of cultured, optic nerve head astrocytes to biomechanical strain, the magnitude and mode of strain based on previously published quantitative models. In this case, astrocytes were subjected to 3 and 12% stretches for either 2 h or 24 h. Proteomic methods included nano-liquid chromatography, tandem mass spectrometry, and iTRAQ labeling. Using controls for both stretch and time, a six-plex iTRAQ liquid chromatography- tandem MS (LC/MS/MS) experiment yielded 573 proteins discovered at a 95% confidence limit. The pathways included transforming growth factor β1, tumor necrosis factor, caspase 3, and tumor protein p53, which have all been implicated in the activation of astrocytes and are believed to play a role in the development of glaucomatous optic neuropathy. Confirmation of the iTRAQ analysis was performed by Western blotting of various proteins of interest including ANXA 4, GOLGA2, and αB-Crystallin. PMID:22126795

The Coming Age of Complete, Accurate, and Ubiquitous Proteomes

DEFF Research Database (Denmark)

Mann, M.; Kulak, N.A.; Nagaraj, N.

2013-01-01

High-resolution mass spectrometry (MS)-based proteomics has progressed tremendously over the years. For model organisms like yeast, we can now quantify complete proteomes in just a few hours. Developments discussed in this Perspective will soon enable complete proteome analysis of mammalian cells...

Proteomic profiling of human plasma exosomes identifies PPARγ as an exosome-associated protein

International Nuclear Information System (INIS)

Looze, Christopher; Yui, David; Leung, Lester; Ingham, Matthew; Kaler, Maryann; Yao, Xianglan; Wu, Wells W.; Shen Rongfong; Daniels, Mathew P.; Levine, Stewart J.

2009-01-01

Exosomes are nanovesicles that are released from cells as a mechanism of cell-free intercellular communication. Only a limited number of proteins have been identified from the plasma exosome proteome. Here, we developed a multi-step fractionation scheme incorporating gel exclusion chromatography, rate zonal centrifugation through continuous sucrose gradients, and high-speed centrifugation to purify exosomes from human plasma. Exosome-associated proteins were separated by SDS-PAGE and 66 proteins were identified by LC-MS/MS, which included both cellular and extracellular proteins. Furthermore, we identified and characterized peroxisome proliferator-activated receptor-γ (PPARγ), a nuclear receptor that regulates adipocyte differentiation and proliferation, as well as immune and inflammatory cell functions, as a novel component of plasma-derived exosomes. Given the important role of exosomes as intercellular messengers, the discovery of PPARγ as a component of human plasma exosomes identifies a potential new pathway for the paracrine transfer of nuclear receptors.

Glycoproteins and Glycosylation Site Assignments in Cereal seed Proteomes

DEFF Research Database (Denmark)

Dedvisitsakul, Plaipol

The study of plant proteomes is important to further the understanding of biological processes and enhance the agronomical and nutritional value of crops and food products. To gain deeper understanding on the proteome level, it is important to characterize posttranslational modifications. Glycosy......The study of plant proteomes is important to further the understanding of biological processes and enhance the agronomical and nutritional value of crops and food products. To gain deeper understanding on the proteome level, it is important to characterize posttranslational modifications...

Mathematical biodescriptors of proteomics maps: background and applications.

Science.gov (United States)

Basak, Subhash C; Gute, Brian D

2008-05-01

This article reviews recent developments in the formulation and application of biodescriptors to characterize proteomics maps. Such biodescriptors can be derived by applying techniques from discrete mathematics (graph theory, linear algebra and information theory). This review focuses on the development of biodescriptors for proteomics maps derived from 2D gel electrophoresis. Preliminary results demonstrated that such descriptors have a reasonable ability to differentiate between proteomics patterns that result from exposure to closely related individual chemicals and complex mixtures, such as the jet fuel JP-8. Further research is required to evaluate the utility of these proteomics-based biodescriptors for drug discovery and predictive toxicology.

Characterization of the porcine synovial fluid proteome and a comparison to the plasma proteome

DEFF Research Database (Denmark)

Bennike, Tue Bjerg; Barnaby, Omar; Steen, Hanno

2015-01-01

Synovial fluid is present in all joint cavities, and protects the articular cartilage surfaces in large by lubricating the joint, thus reducing friction. Several studies have described changes in the protein composition of synovial fluid in patients with joint disease. However, the protein concen...... data used in the method optimization, human plasma proteomics data, and search results, have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD000935....

Identification Of Protein Vaccine Candidates Using Comprehensive Proteomic Analysis Strategies

Science.gov (United States)

2007-12-01

that fascinating fungus known as Coccidioides. I also want to thank the UA Mass Spectrometry Facility and the UA Proteomics Consortium, especially...W. & N. N. Kav. 2006. The proteome of the phytopathogenic fungus Sclerotinia sclerotiorum. Proteomics 6: 5995-6007. 127. de Godoy, L. M., J. V...IDENTIFICATION OF PROTEIN VACCINE CANDIDATES USING COMPREHENSIVE PROTEOMIC ANALYSIS STRATEGIES by James G. Rohrbough

Serum proteome profiling in canine idiopathic dilated cardiomyopathy using TMT-based quantitative proteomics approach.

Science.gov (United States)

Bilić, Petra; Guillemin, Nicolas; Kovačević, Alan; Beer Ljubić, Blanka; Jović, Ines; Galan, Asier; Eckersall, Peter David; Burchmore, Richard; Mrljak, Vladimir

2018-05-15

Idiopathic dilated cardiomyopathy (iDCM) is a primary myocardial disorder with an unknown aetiology, characterized by reduced contractility and ventricular dilation of the left or both ventricles. Naturally occurring canine iDCM was used herein to identify serum proteomic signature of the disease compared to the healthy state, providing an insight into underlying mechanisms and revealing proteins with biomarker potential. To achieve this, we used high-throughput label-based quantitative LC-MS/MS proteomics approach and bioinformatics analysis of the in silico inferred interactome protein network created from the initial list of differential proteins. To complement the proteomic analysis, serum biochemical parameters and levels of know biomarkers of cardiac function were measured. Several proteins with biomarker potential were identified, such as inter-alpha-trypsin inhibitor heavy chain H4, microfibril-associated glycoprotein 4 and apolipoprotein A-IV, which were validated using an independent method (Western blotting) and showed high specificity and sensitivity according to the receiver operating characteristic curve analysis. Bioinformatics analysis revealed involvement of different pathways in iDCM, such as complement cascade activation, lipoprotein particles dynamics, elastic fibre formation, GPCR signalling and respiratory electron transport chain. Idiopathic dilated cardiomyopathy is a severe primary myocardial disease of unknown cause, affecting both humans and dogs. This study is a contribution to the canine heart disease research by means of proteomic and bioinformatic state of the art analyses, following similar approach in human iDCM research. Importantly, we used serum as non-invasive and easily accessible biological source of information and contributed to the scarce data on biofluid proteome research on this topic. Bioinformatics analysis revealed biological pathways modulated in canine iDCM with potential of further targeted research. Also, several

Proteomics: Protein Identification Using Online Databases

Science.gov (United States)

Eurich, Chris; Fields, Peter A.; Rice, Elizabeth

2012-01-01

Proteomics is an emerging area of systems biology that allows simultaneous study of thousands of proteins expressed in cells, tissues, or whole organisms. We have developed this activity to enable high school or college students to explore proteomic databases using mass spectrometry data files generated from yeast proteins in a college laboratory…

Modification-specific proteomics in plant biology

DEFF Research Database (Denmark)

Ytterberg, A Jimmy; Jensen, Ole N

2010-01-01

and proteomics. In general, methods for PTM characterization are developed to study yeast and mammalian biology and later adopted to investigate plants. Our point of view is that it is advantageous to enrich for PTMs on the peptide level as part of a quantitative proteomics strategy to not only identify the PTM...

Eosinophilic Otitis Media: the Aftermath of Eosinophil Extracellular Trap Cell Death.

Science.gov (United States)

Ueki, Shigeharu; Ohta, Nobuo; Takeda, Masahide; Konno, Yasunori; Hirokawa, Makoto

2017-05-01

Eosinophilic otitis media (EOM) is a refractory disease characterized by the accumulation of eosinophils in middle ear effusion and mucosa. We summarize current knowledge regarding the clinical characteristics and management of EOM. Although eosinophil activation in inflamed foci is involved in the pathogenesis of EOM, little is known about the fate of the eosinophils and aftermath of their cell death. We discuss the possibility that eosinophils undergo non-apoptotic cell death that worsens tissue damage and increases effusion viscosity. Unlike chronic otitis media, EOM is strongly associated with an allergic background. Corticosteroids are currently the only effective pharmacological treatment, and surgical intervention is often required. Mucosal eosinophils infiltrate extensively into the middle ear cavity where they are stimulated by locally produced activators including interleukin-5 and eotaxin. The eosinophils undergo cytolysis in the effusion, which represents a major fate of activated eosinophils in vivo. Recent data revealed cytolysis could be renamed as extracellular trap cell death (ETosis). ETosis represents suicidal cell death involving total cell degranulation and development of sticky chromatin structures (extracellular traps (ETs)). The characteristics of eosinophil- and neutrophil-derived ET polymers might contribute to the difference in viscosity of secretions between EOM and common chronic otitis media. The extracellular products remaining after eosinophil ETosis are an important aspect of EOM pathology. The concept of ETosis also has novel implications for potential therapeutic modalities in various eosinophilic disorders.

Levels of Circulating MMCN-151, a Degradation Product of Mimecan, Reflect Pathological Extracellular Matrix Remodeling in Apolipoprotein E Knockout Mice

DEFF Research Database (Denmark)

Barascuk, N; Vassiliadis, E; Zheng, Qiuju

2011-01-01

Arterial extracellular matrix (ECM) remodeling by matrix metalloproteinases (MMPs) is one of the major hallmarks of atherosclerosis. Mimecan, also known as osteoglycin has been implicated in the integrity of the ECM. This study assessed the validity of an enzyme-linked immunosorbent assay (ELISA...

The colostrum proteome, ruminant nutrition and immunity: a review.

Science.gov (United States)

Hernández-Castellano, Lorenzo E; Almeida, Andrè M; Castro, Noemi; Argüello, Anastasio

2014-02-01

In this review authors address colostrum proteins implications in different domestic ruminant species. The colostrogenesis process and how different factors, such as litter size or nutrition during gestation can alter the different components concentrations in colostrum are also reviewed. The different colostrum fractions will be described, focusing on high and low abundant proteins. This review describes the major function of such proteins and their role on the passive immune transfer and nutrition in the newborn animal. It will be also performed a comprehensive review on different techniques and commercial kits available for high abundant protein depletion in colostrum. We will finally focus on how proteomics has been used to address this issue and how it can contribute to the major questions about colostrum associated immunology.

Integrative Analysis of Subcellular Quantitative Proteomics Studies Reveals Functional Cytoskeleton Membrane-Lipid Raft Interactions in Cancer.

Science.gov (United States)

Shah, Anup D; Inder, Kerry L; Shah, Alok K; Cristino, Alexandre S; McKie, Arthur B; Gabra, Hani; Davis, Melissa J; Hill, Michelle M

2016-10-07

Lipid rafts are dynamic membrane microdomains that orchestrate molecular interactions and are implicated in cancer development. To understand the functions of lipid rafts in cancer, we performed an integrated analysis of quantitative lipid raft proteomics data sets modeling progression in breast cancer, melanoma, and renal cell carcinoma. This analysis revealed that cancer development is associated with increased membrane raft-cytoskeleton interactions, with ∼40% of elevated lipid raft proteins being cytoskeletal components. Previous studies suggest a potential functional role for the raft-cytoskeleton in the action of the putative tumor suppressors PTRF/Cavin-1 and Merlin. To extend the observation, we examined lipid raft proteome modulation by an unrelated tumor suppressor opioid binding protein cell-adhesion molecule (OPCML) in ovarian cancer SKOV3 cells. In agreement with the other model systems, quantitative proteomics revealed that 39% of OPCML-depleted lipid raft proteins are cytoskeletal components, with microfilaments and intermediate filaments specifically down-regulated. Furthermore, protein-protein interaction network and simulation analysis showed significantly higher interactions among cancer raft proteins compared with general human raft proteins. Collectively, these results suggest increased cytoskeleton-mediated stabilization of lipid raft domains with greater molecular interactions as a common, functional, and reversible feature of cancer cells.

Biomarker discovery in mass spectrometry-based urinary proteomics.

Science.gov (United States)

Thomas, Samuel; Hao, Ling; Ricke, William A; Li, Lingjun

2016-04-01

Urinary proteomics has become one of the most attractive topics in disease biomarker discovery. MS-based proteomic analysis has advanced continuously and emerged as a prominent tool in the field of clinical bioanalysis. However, only few protein biomarkers have made their way to validation and clinical practice. Biomarker discovery is challenged by many clinical and analytical factors including, but not limited to, the complexity of urine and the wide dynamic range of endogenous proteins in the sample. This article highlights promising technologies and strategies in the MS-based biomarker discovery process, including study design, sample preparation, protein quantification, instrumental platforms, and bioinformatics. Different proteomics approaches are discussed, and progresses in maximizing urinary proteome coverage and standardization are emphasized in this review. MS-based urinary proteomics has great potential in the development of noninvasive diagnostic assays in the future, which will require collaborative efforts between analytical scientists, systems biologists, and clinicians. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Review and Database of Snake Venom Proteomes.

Science.gov (United States)

Tasoulis, Theo; Isbister, Geoffrey K

2017-09-18

Advances in the last decade combining transcriptomics with established proteomics methods have made possible rapid identification and quantification of protein families in snake venoms. Although over 100 studies have been published, the value of this information is increased when it is collated, allowing rapid assimilation and evaluation of evolutionary trends, geographical variation, and possible medical implications. This review brings together all compositional studies of snake venom proteomes published in the last decade. Compositional studies were identified for 132 snake species: 42 from 360 (12%) Elapidae (elapids), 20 from 101 (20%) Viperinae (true vipers), 65 from 239 (27%) Crotalinae (pit vipers), and five species of non-front-fanged snakes. Approximately 90% of their total venom composition consisted of eight protein families for elapids, 11 protein families for viperines and ten protein families for crotalines. There were four dominant protein families: phospholipase A₂s (the most common across all front-fanged snakes), metalloproteases, serine proteases and three-finger toxins. There were six secondary protein families: cysteine-rich secretory proteins, l-amino acid oxidases, kunitz peptides, C-type lectins/snaclecs, disintegrins and natriuretic peptides. Elapid venoms contained mostly three-finger toxins and phospholipase A₂s and viper venoms metalloproteases, phospholipase A₂s and serine proteases. Although 63 protein families were identified, more than half were present in <5% of snake species studied and always in low abundance. The importance of these minor component proteins remains unknown.

Data in support of the identification of neuronal and astrocyte proteins interacting with extracellularly applied oligomeric and fibrillar α-synuclein assemblies by mass spectrometry

Directory of Open Access Journals (Sweden)

Amulya Nidhi Shrivastava

2016-06-01

Full Text Available α-Synuclein (α-syn is the principal component of Lewy bodies, the pathophysiological hallmark of individuals affected by Parkinson disease (PD. This neuropathologic form of α-syn contributes to PD progression and propagation of α-syn assemblies between neurons. The data we present here support the proteomic analysis used to identify neuronal proteins that specifically interact with extracellularly applied oligomeric or fibrillar α-syn assemblies (conditions 1 and 2, respectively (doi: 10.15252/embj.201591397 [1]. α-syn assemblies and their cellular partner proteins were pulled down from neuronal cell lysed shortly after exposure to exogenous α-syn assemblies and the associated proteins were identified by mass spectrometry using a shotgun proteomic-based approach. We also performed experiments on pure cultures of astrocytes to identify astrocyte-specific proteins interacting with oligomeric or fibrillar α-syn (conditions 3 and 4, respectively. For each condition, proteins interacting selectively with α-syn assemblies were identified by comparison to proteins pulled-down from untreated cells used as controls. The mass spectrometry data, the database search and the peak lists have been deposited to the ProteomeXchange Consortium database via the PRIDE partner repository with the dataset identifiers PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD002256 to PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD002263 and doi: 10.6019/http://www.ebi.ac.uk/pride/archive/projects/PXD002256 to 10.6019/http://www.ebi.ac.uk/pride/archive/projects/PXD002263.

Plant Abiotic Stress Proteomics: The Major Factors Determining Alterations in Cellular Proteome

Science.gov (United States)

Kosová, Klára; Vítámvás, Pavel; Urban, Milan O.; Prášil, Ilja T.; Renaut, Jenny

2018-01-01

HIGHLIGHTS: Major environmental and genetic factors determining stress-related protein abundance are discussed.Major aspects of protein biological function including protein isoforms and PTMs, cellular localization and protein interactions are discussed.Functional diversity of protein isoforms and PTMs is discussed. Abiotic stresses reveal profound impacts on plant proteomes including alterations in protein relative abundance, cellular localization, post-transcriptional and post-translational modifications (PTMs), protein interactions with other protein partners, and, finally, protein biological functions. The main aim of the present review is to discuss the major factors determining stress-related protein accumulation and their final biological functions. A dynamics of stress response including stress acclimation to altered ambient conditions and recovery after the stress treatment is discussed. The results of proteomic studies aimed at a comparison of stress response in plant genotypes differing in stress adaptability reveal constitutively enhanced levels of several stress-related proteins (protective proteins, chaperones, ROS scavenging- and detoxification-related enzymes) in the tolerant genotypes with respect to the susceptible ones. Tolerant genotypes can efficiently adjust energy metabolism to enhanced needs during stress acclimation. Stress tolerance vs. stress susceptibility are relative terms which can reflect different stress-coping strategies depending on the given stress treatment. The role of differential protein isoforms and PTMs with respect to their biological functions in different physiological constraints (cellular compartments and interacting partners) is discussed. The importance of protein functional studies following high-throughput proteome analyses is presented in a broader context of plant biology. In summary, the manuscript tries to provide an overview of the major factors which have to be considered when interpreting data from proteomic

Proteomic approach to nanotoxicity.

Science.gov (United States)

Matysiak, Magdalena; Kapka-Skrzypczak, Lucyna; Brzóska, Kamil; Gutleb, Arno C; Kruszewski, Marcin

2016-03-30

In recent years a large number of engineered nanomaterials (NMs) have been developed with promising technical benefits for consumers and medical appliances. In addition to already known potentially advantageous biological properties (antibiotic, antifungal and antiviral activity) of NMs, many new medical applications of NMs are foreseen, such as drug carriers, contrast agents, radiopharmaceuticals and many others. However, there is increasing concern about potential environmental and health effects due to NMs exposure. An increasing body of evidence suggests that NMs may trigger undesirable hazardous interactions with biological systems with potential to generate harmful effects. In this review we summarized a current state of knowledge on the proteomics approaches to nanotoxicity, including protein corona formation, in vitro and in vivo effects of exposure to NMs on proteome of different classes of organisms, from bacteria and plants to mammals. The effects of NMs on the proteome of environmentally relevant organisms are also described. Despite the benefit that development of nanotechnology may bring to the society, there are still major gaps of knowledge on the influence of nanomaterials on human health and the environment. Thus, it seems necessary to conduct further interdisciplinary research to fill the knowledge gaps in NM toxicity, using more holistic approaches than offered by conventional biological techniques. “OMICS” techniques will certainly help researchers in this field. In this paper we summarized the current stage of knowledge of the effects of nanoparticles on the proteome of different organisms, including those commonly used as an environmentally relevant indicator organisms.

The quest of the human proteome and the missing proteins: digging deeper.

Science.gov (United States)

Reddy, Panga Jaipal; Ray, Sandipan; Srivastava, Sanjeeva

2015-05-01

Given the diverse range of transcriptional and post-transcriptional mechanisms of gene regulation, the estimates of the human proteome is likely subject to scientific surprises as the field of proteomics has gained momentum worldwide. In this regard, the establishment of the "Human Proteome Draft" using high-resolution mass spectrometry (MS), tissue microarrays, and immunohistochemistry by three independent research groups (laboratories of Pandey, Kuster, and Uhlen) accelerated the pace of proteomics research. The Chromosome Centric Human Proteome Project (C-HPP) has taken initiative towards the completion of the Human Proteome Project (HPP) so as to understand the proteomics correlates of common complex human diseases and biological diversity, not to mention person-to-person and population differences in response to drugs, nutrition, vaccines, and other health interventions and host-environment interactions. Although high-resolution MS-based and antibody microarray approaches have shown enormous promises, we are still unable to map the whole human proteome due to the presence of numerous "missing proteins." In December 2014, at the Indian Institute of Technology Bombay, Mumbai the 6(th) Annual Meeting of the Proteomics Society, India (PSI) and the International Proteomics Conference was held. As part of this interdisciplinary summit, a panel discussion session on "The Quest of the Human Proteome and Missing Proteins" was organized. Eminent scientists in the field of proteomics and systems biology, including Akhilesh Pandey, Gilbert S. Omenn, Mark S. Baker, and Robert L. Mortiz, shed light on different aspects of the human proteome drafts and missing proteins. Importantly, the possible reasons for the "missing proteins" in shotgun MS workflow were identified and debated by experts as low tissue expression, lack of enzymatic digestion site, or protein lost during extraction, among other contributing factors. To capture the missing proteins, the experts' collective

Clinical proteomics: Current status, challenges, and future perspectives

Directory of Open Access Journals (Sweden)

Shyh-Horng Chiou

2011-01-01

Full Text Available This account will give an overview and evaluation of the current advances in mass spectrometry (MS-based proteomics platforms and technology. A general review of some background information concerning the application of these methods in the characterization of molecular sizes and related protein expression profiles associated with different types of cells under varied experimental conditions will be presented. It is intended to provide a concise and succinct overview to those clinical researchers first exposed to this foremost powerful methodology in modern life sciences of postgenomic era. Proteomic characterization using highly sophisticated and expensive instrumentation of MS has been used to characterize biological samples of complex protein mixtures with vastly different protein structure and composition. These systems are then used to highlight the versatility and potential of the MS-based proteomic strategies for facilitating protein expression analysis of various disease-related organisms or tissues of interest. Major MS-based strategies reviewed herein include (1 matrix-assisted laser desorption ionization-MS and electron-spray ionization proteomics; (2 one-dimensional or two-dimensional gel-based proteomics; (3 gel-free shotgun proteomics in conjunction with liquid chromatography/tandem MS; (4 Multiple reaction monitoring coupled tandem MS quantitative proteomics and; (5 Phosphoproteomics based on immobilized metal affinity chromatography and liquid chromatography-MS/MS.

A community proposal to integrate proteomics activities in ELIXIR.

Science.gov (United States)

Vizcaíno, Juan Antonio; Walzer, Mathias; Jiménez, Rafael C; Bittremieux, Wout; Bouyssié, David; Carapito, Christine; Corrales, Fernando; Ferro, Myriam; Heck, Albert J R; Horvatovich, Peter; Hubalek, Martin; Lane, Lydie; Laukens, Kris; Levander, Fredrik; Lisacek, Frederique; Novak, Petr; Palmblad, Magnus; Piovesan, Damiano; Pühler, Alfred; Schwämmle, Veit; Valkenborg, Dirk; van Rijswijk, Merlijn; Vondrasek, Jiri; Eisenacher, Martin; Martens, Lennart; Kohlbacher, Oliver

2017-01-01

Computational approaches have been major drivers behind the progress of proteomics in recent years. The aim of this white paper is to provide a framework for integrating computational proteomics into ELIXIR in the near future, and thus to broaden the portfolio of omics technologies supported by this European distributed infrastructure. This white paper is the direct result of a strategy meeting on 'The Future of Proteomics in ELIXIR' that took place in March 2017 in Tübingen (Germany), and involved representatives of eleven ELIXIR nodes. These discussions led to a list of priority areas in computational proteomics that would complement existing activities and close gaps in the portfolio of tools and services offered by ELIXIR so far. We provide some suggestions on how these activities could be integrated into ELIXIR's existing platforms, and how it could lead to a new ELIXIR use case in proteomics. We also highlight connections to the related field of metabolomics, where similar activities are ongoing. This white paper could thus serve as a starting point for the integration of computational proteomics into ELIXIR. Over the next few months we will be working closely with all stakeholders involved, and in particular with other representatives of the proteomics community, to further refine this paper.

"Does understanding the brain need proteomics and does understanding proteomics need brains?"--Second HUPO HBPP Workshop hosted in Paris.

Science.gov (United States)

Hamacher, Michael; Klose, Joachim; Rossier, Jean; Marcus, Katrin; Meyer, Helmut E

2004-07-01

The second Human Brain Proteome Project (HBPP) Workshop of the Human Proteome Organisation (HUPO) took place at the Ecole Supérieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI) from April 23-24, 2004. During two days, more than 70 attendees from Europe, Asia and the US came together to decide basic strategic approaches, standards and the beginning of a pilot phase prior to further studies of the human brain proteome. The international consortium presented the technological and scientific portfolio and scheduled the time table for the next year.

Activation of retinal glial (Müller cells by extracellular ATP induces pronounced increases in extracellular H+ flux.

Directory of Open Access Journals (Sweden)

Boriana K Tchernookova

Full Text Available Small alterations in extracellular acidity are potentially important modulators of neuronal signaling within the vertebrate retina. Here we report a novel extracellular acidification mechanism mediated by glial cells in the retina. Using self-referencing H+-selective microelectrodes to measure extracellular H+ fluxes, we show that activation of retinal Müller (glial cells of the tiger salamander by micromolar concentrations of extracellular ATP induces a pronounced extracellular H+ flux independent of bicarbonate transport. ADP, UTP and the non-hydrolyzable analog ATPγs at micromolar concentrations were also potent stimulators of extracellular H+ fluxes, but adenosine was not. The extracellular H+ fluxes induced by ATP were mimicked by the P2Y1 agonist MRS 2365 and were significantly reduced by the P2 receptor blockers suramin and PPADS, suggesting activation of P2Y receptors. Bath-applied ATP induced an intracellular rise in calcium in Müller cells; both the calcium rise and the extracellular H+ fluxes were significantly attenuated when calcium re-loading into the endoplasmic reticulum was inhibited by thapsigargin and when the PLC-IP3 signaling pathway was disrupted with 2-APB and U73122. The anion transport inhibitor DIDS also markedly reduced the ATP-induced increase in H+ flux while SITS had no effect. ATP-induced H+ fluxes were also observed from Müller cells isolated from human, rat, monkey, skate and lamprey retinae, suggesting a highly evolutionarily conserved mechanism of potential general importance. Extracellular ATP also induced significant increases in extracellular H+ flux at the level of both the outer and inner plexiform layers in retinal slices of tiger salamander which was significantly reduced by suramin and PPADS. We suggest that the novel H+ flux mediated by ATP-activation of Müller cells and of other glia as well may be a key mechanism modulating neuronal signaling in the vertebrate retina and throughout the brain.

Pre-fractionation strategies to resolve pea (Pisum sativum sub-proteomes

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Claudia Nicole Meisrimler

2015-10-01

Full Text Available Legumes are important crop plants and pea (Pisum sativum L. has been investigated as a model with respect to several physiological aspects. The sequencing of the pea genome has not been completed. Therefore, proteomic approaches are currently limited. Nevertheless, the increasing numbers of available EST-databases as well as the high homology of the pea and medicago genome (Medicago truncatula G. allow the successful identification of proteins. Due to the un-sequenced pea genome, pre-fractionation approaches have been used in pea proteomic surveys in the past. Aside from a number of selective proteome studies on crude extracts and the chloroplast, few studies have targeted other components such as the pea secretome, an important sub-proteome of interest due to its role in abiotic and biotic stress processes. The secretome itself can be further divided into different sub-proteomes (plasma membrane, apoplast, cell wall proteins. Cell fractionation in combination with different gel-electrophoresis, chromatography methods and protein identification by mass spectrometry are important partners to gain insight into pea sub-proteomes, post-translational modifications and protein functions. Overall, pea proteomics needs to link numerous existing physiological and biochemical data to gain further insight into adaptation processes, which play important roles in field applications. Future developments and directions in pea proteomics are discussed.

Proteomic and metabolomic approaches to biomarker discovery

CERN Document Server

Issaq, Haleem J

2013-01-01

Proteomic and Metabolomic Approaches to Biomarker Discovery demonstrates how to leverage biomarkers to improve accuracy and reduce errors in research. Disease biomarker discovery is one of the most vibrant and important areas of research today, as the identification of reliable biomarkers has an enormous impact on disease diagnosis, selection of treatment regimens, and therapeutic monitoring. Various techniques are used in the biomarker discovery process, including techniques used in proteomics, the study of the proteins that make up an organism, and metabolomics, the study of chemical fingerprints created from cellular processes. Proteomic and Metabolomic Approaches to Biomarker Discovery is the only publication that covers techniques from both proteomics and metabolomics and includes all steps involved in biomarker discovery, from study design to study execution.  The book describes methods, and presents a standard operating procedure for sample selection, preparation, and storage, as well as data analysis...

Characterization of individual mouse cerebrospinal fluid proteomes

Energy Technology Data Exchange (ETDEWEB)

Smith, Jeffrey S.; Angel, Thomas E.; Chavkin, Charles; Orton, Daniel J.; Moore, Ronald J.; Smith, Richard D.

2014-03-20

Analysis of cerebrospinal fluid (CSF) offers key insight into the status of the central nervous system. Characterization of murine CSF proteomes can provide a valuable resource for studying central nervous system injury and disease in animal models. However, the small volume of CSF in mice has thus far limited individual mouse proteome characterization. Through non-terminal CSF extractions in C57Bl/6 mice and high-resolution liquid chromatography-mass spectrometry analysis of individual murine samples, we report the most comprehensive proteome characterization of individual murine CSF to date. Utilizing stringent protein inclusion criteria that required the identification of at least two unique peptides (1% false discovery rate at the peptide level) we identified a total of 566 unique proteins, including 128 proteins from three individual CSF samples that have been previously identified in brain tissue. Our methods and analysis provide a mechanism for individual murine CSF proteome analysis.

Inhibiting extracellular matrix metalloproteinase inducer maybe beneficial for diminishing the atherosclerotic plaque instability

Directory of Open Access Journals (Sweden)

Xie S

2009-01-01

Full Text Available Atherosclerotic plaque rupture and local thrombosis activation in the artery cause acute serious incidents such as acute coronary syndrome and stroke. The exact mechanism of plaque rupture remains unclear but excessive degradation of the extracellular matrix scaffold by matrix-degrading metalloproteinases (MMPs has been implicated as one of the major molecular mechanisms in this process. Convincing evidence is available to prove that extracellular matrix metalloproteinase inducer (EMMPRIN induces MMP expression and is involved in the inflammatory responses in the artery wall. The inflammation and MMPs have been shown to play a critical role for atherosclerotic lesion development and progression. More recent data showed that increased EMMPRIN expression was associated with vulnerable atherosclerotic lesions. Therefore, we speculate that EMMPRIN may be pivotal for atherosclerotic plaque instability, and hence inhibition of EMMPRIN expression could be a promising approach for the prevention or treatment of atheroma instability.

Venomous snakes of Costa Rica: biological and medical implications of their venom proteomic profiles analyzed through the strategy of snake venomics.

Science.gov (United States)

Lomonte, Bruno; Fernández, Julián; Sanz, Libia; Angulo, Yamileth; Sasa, Mahmood; Gutiérrez, José María; Calvete, Juan J

2014-06-13

In spite of its small territory of ~50,000km(2), Costa Rica harbors a remarkably rich biodiversity. Its herpetofauna includes 138 species of snakes, of which sixteen pit vipers (family Viperidae, subfamily Crotalinae), five coral snakes (family Elapidae, subfamily Elapinae), and one sea snake (Family Elapidae, subfamily Hydrophiinae) pose potential hazards to human and animal health. In recent years, knowledge on the composition of snake venoms has expanded dramatically thanks to the development of increasingly fast and sensitive analytical techniques in mass spectrometry and separation science applied to protein characterization. Among several analytical strategies to determine the overall protein/peptide composition of snake venoms, the methodology known as 'snake venomics' has proven particularly well suited and informative, by providing not only a catalog of protein types/families present in a venom, but also a semi-quantitative estimation of their relative abundances. Through a collaborative research initiative between Instituto de Biomedicina de Valencia (IBV) and Instituto Clodomiro Picado (ICP), this strategy has been applied to the study of venoms of Costa Rican snakes, aiming to obtain a deeper knowledge on their composition, geographic and ontogenic variations, relationships to taxonomy, correlation with toxic activities, and discovery of novel components. The proteomic profiles of venoms from sixteen out of the 22 species within the Viperidae and Elapidae families found in Costa Rica have been reported so far, and an integrative view of these studies is hereby presented. In line with other venomic projects by research groups focusing on a wide variety of snakes around the world, these studies contribute to a deeper understanding of the biochemical basis for the diverse toxic profiles evolved by venomous snakes. In addition, these studies provide opportunities to identify novel molecules of potential pharmacological interest. Furthermore, the

Extracellular sphingosine-1-phosphate: a novel actor in human glioblastoma stem cell survival.

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Elena Riccitelli

Full Text Available Glioblastomas are the most frequent and aggressive intracranial neoplasms in humans, and despite advances and the introduction of the alkylating agent temozolomide in therapy have improved patient survival, resistance mechanisms limit benefits. Recent studies support that glioblastoma stem-like cells (GSCs, a cell subpopulation within the tumour, are involved in the aberrant expansion and therapy resistance properties of glioblastomas, through still unclear mechanisms. Emerging evidence suggests that sphingosine-1-phosphate (S1P a potent onco-promoter able to act as extracellular signal, favours malignant and chemoresistance properties in GSCs. Notwithstanding, the origin of S1P in the GSC environment remains unknown. We investigated S1P metabolism, release, and role in cell survival properties of GSCs isolated from either U87-MG cell line or a primary culture of human glioblastoma. We show that both GSC models, grown as neurospheres and expressing GSC markers, are resistant to temozolomide, despite not expressing the DNA repair protein MGMT, a major contributor to temozolomide-resistance. Pulse experiments with labelled sphingosine revealed that both GSC types are able to rapidly phosphorylate the long-chain base, and that the newly produced S1P is efficiently degraded. Of relevance, we found that S1P was present in GSC extracellular medium, its level being significantly higher than in U87-MG cells, and that the extracellular/intracellular ratio of S1P was about ten-fold higher in GSCs. The activity of sphingosine kinases was undetectable in GSC media, suggesting that mechanisms of S1P transport to the extracellular environment are constitutive in GSCs. In addition we found that an inhibitor of S1P biosynthesis made GSCs sensitive to temozolomide (TMZ, and that exogenous S1P reverted this effect, thus involving extracellular S1P as a GSC survival signal in TMZ resistance. Altogether our data implicate for the first time GSCs as a pivotal source

Antibody Binding Alters the Characteristics and Contents of Extracellular Vesicles Released by Histoplasma capsulatum

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Baltazar, Ludmila M.; Nakayasu, Ernesto S.; Sobreira, Tiago; Choi, Hyungwon; Casadevall, Arturo; Nimrichter, Leonardo; Nosanchuk, Joshua D.

2016-03-30

Histoplasma capsulatumproduces extracellular vesicles containing virulence-associated molecules capable of modulating host machinery, benefiting the pathogen. Treatment ofH. capsulatumcells with monoclonal antibodies (MAbs) can change the outcome of infection in mice. We evaluated the sizes, enzymatic contents, and proteomic profiles of the vesicles released by fungal cells treated with either protective MAb 6B7 (IgG1) or nonprotective MAb 7B6 (IgG2b), both of which bindH. capsulatumheat shock protein 60 (Hsp60). Our results showed that treatment with either MAb was associated with changes in size and vesicle loading. MAb treatments reduced vesicle phosphatase and catalase activities compared to those of vesicles from untreated controls. We identified 1,125 proteins in vesicles, and 250 of these manifested differences in abundance relative to that of proteins in vesicles isolated from yeast cells exposed to Hsp60-binding MAbs, indicating that surface binding of fungal cells by MAbs modified protein loading in the vesicles. The abundance of upregulated proteins in vesicles upon MAb 7B6 treatment was 44.8% of the protein quantities in vesicles from fungal cells treated with MAb 6B7. Analysis of orthologous proteins previously identified in vesicles from other fungi showed that different ascomycete fungi have similar proteins in their extracellular milieu, many of which are associated with virulence. Our results demonstrate that antibody binding can modulate fungal cell responses, resulting in differential loading of vesicles, which could alter fungal cell susceptibility to host defenses. This finding provides additional evidence that antibody binding modulates microbial physiology and suggests a new function for specific immunoglobulins through alterations of fungal secretion.

IMPORTANCEDiverse fungal species release extracellular vesicles, indicating that this is a

Inspection, visualisation and analysis of quantitative proteomics data

OpenAIRE

Gatto, Laurent

2016-01-01

Material Quantitative Proteomics and Data Analysis Course. 4 - 5 April 2016, Queen Hotel, Chester, UK Table D - Inspection, visualisation and analysis of quantitative proteomics data, Laurent Gatto (University of Cambridge)

Revisiting biomarker discovery by plasma proteomics

DEFF Research Database (Denmark)

Geyer, Philipp E; Holdt, Lesca M; Teupser, Daniel

2017-01-01

slow rate. As described in this review, mass spectrometry (MS)-based proteomics has become a powerful technology in biological research and it is now poised to allow the characterization of the plasma proteome in great depth. Previous "triangular strategies" aimed at discovering single biomarker...

Expanding the bovine milk proteome through extensive fractionation.

Science.gov (United States)

Nissen, Asger; Bendixen, Emøke; Ingvartsen, Klaus Lønne; Røntved, Christine Maria

2013-01-01

Bovine milk is an agricultural product of tremendous value worldwide. It contains proteins, fat, lactose, vitamins, and minerals. It provides nutrition and immunological protection (e.g., in the gastrointestinal tract) to the newborn and young calf. It also forms an important part of human nutrition. The repertoire of proteins in milk (i.e., its proteome) is vast and complex. The milk proteome can be described in detail by mass spectrometry-based proteomics. However, the high concentration of dominating proteins in milk reduces mass spectrometry detection sensitivity and limits detection of low abundant proteins. Further, the general health and udder health of the dairy cows delivering the milk may influence the composition of the milk proteome. To gain a more exhaustive and true picture of the milk proteome, we performed an extensive preanalysis fractionation of raw composite milk collected from documented healthy cows in early lactation. Four simple and industrially applicable techniques exploring the physical and chemical properties of milk, including acidification, filtration, and centrifugation, were used for separation of the proteins. This resulted in 5 different fractions, whose content of proteins were compared with the proteins of nonfractionated milk using 2-dimensional liquid chromatography tandem mass spectrometry analysis. To validate the proteome analysis, spectral counts and ELISA were performed on 7 proteins using the ELISA for estimation of the detection sensitivity limit of the 2-dimensional liquid chromatography tandem mass spectrometry analysis. Each fractionation technique resulted in identification of a unique subset of proteins. However, high-speed centrifugation of milk to whey was by far the best method to achieve high and repeatable proteome coverage. The total number of milk proteins initially detected in nonfractionated milk and the fractions were 635 in 2 replicates. Removal of dominant proteins and filtering for redundancy across the

Comprehensive proteomic analysis of human pancreatic juice

DEFF Research Database (Denmark)

Grønborg, Mads; Bunkenborg, Jakob; Kristiansen, Troels Zakarias

2004-01-01

Proteomic technologies provide an excellent means for analysis of body fluids for cataloging protein constituents and identifying biomarkers for early detection of cancers. The biomarkers currently available for pancreatic cancer, such as CA19-9, lack adequate sensitivity and specificity...... contributing to late diagnosis of this deadly disease. In this study, we carried out a comprehensive characterization of the "pancreatic juice proteome" in patients with pancreatic adenocarcinoma. Pancreatic juice was first fractionated by 1-dimensional gel electrophoresis and subsequently analyzed by liquid...... in this study could be directly assessed for their potential as biomarkers for pancreatic cancer by quantitative proteomics methods or immunoassays....

Comparative proteomics of mitosis and meiosis in Saccharomyces cerevisiae.

Science.gov (United States)

Kumar, Ravinder; Dhali, Snigdha; Srikanth, Rapole; Ghosh, Santanu Kumar; Srivastava, Sanjeeva

2014-09-23

Precise and timely segregation of genetic material and conservation of ploidy are the two foremost requirements for survival of a eukaryotic organism. Two highly regulated cell division processes, namely mitosis and meiosis are central to achieve this objective. The modes of chromosome segregation are distinct in these two processes that generate progeny cells of equal ploidy and half the ploidy in mitosis and meiosis, respectively. Additionally, the nutritional requirement and intracellular processing of biological cue also differ in these two processes. From this, it can be envisaged that proteome of mitotic and meiotic cells will differ significantly. Therefore, identification of proteins that differ in their level of expression between mitosis and meiosis would further reveal the mechanistic detail of these processes. In the present study, we have investigated the protein expression profile of mitosis and meiosis by comparing proteome of budding yeast cultures arrested at mitotic metaphase and metaphase-I of meiosis using proteomic approach. Approximately 1000 and 2000 protein spots were visualized on 2-DE and 2D-DIGE gels respectively, out of which 14 protein spots were significant in 2-DE and 22 in 2D-DIGE (pmitosis, an up-regulation of actin cytoskeleton and its negative regulator occurs in meiosis. Mitosis and meiosis are two different types of cell division cycles with entirely different outcomes with definite biological implication for almost all eukaryotic species. In this work, we investigated, for the first time, the differential proteomic profile of Saccharomyces cerevisiae culture arrested at mitotic metaphase (M) and metaphase-I (MI) of meiosis using 2-DE and 2D-DIGE. Our findings of up-regulation of actin and its negative regulator cofilin during meiosis suggest that the rate of actin cytoskeleton turnover is more in meiosis and actin cytoskeleton may play more crucial role during meiosis compared to mitosis. Present study also suggests that actin

Liver proteomics in progressive alcoholic steatosis

Energy Technology Data Exchange (ETDEWEB)

Fernando, Harshica [Department of Pathology, The University of Texas Medical Branch, Galveston, TX 77555 (United States); Wiktorowicz, John E.; Soman, Kizhake V. [Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, TX 77555 (United States); Kaphalia, Bhupendra S.; Khan, M. Firoze [Department of Pathology, The University of Texas Medical Branch, Galveston, TX 77555 (United States); Shakeel Ansari, G.A., E-mail: sansari@utmb.edu [Department of Pathology, The University of Texas Medical Branch, Galveston, TX 77555 (United States)

2013-02-01

Fatty liver is an early stage of alcoholic and nonalcoholic liver disease (ALD and NALD) that progresses to steatohepatitis and other irreversible conditions. In this study, we identified proteins that were differentially expressed in the livers of rats fed 5% ethanol in a Lieber–DeCarli diet daily for 1 and 3 months by discovery proteomics (two-dimensional gel electrophoresis and mass spectrometry) and non-parametric modeling (Multivariate Adaptive Regression Splines). Hepatic fatty infiltration was significantly higher in ethanol-fed animals as compared to controls, and more pronounced at 3 months of ethanol feeding. Discovery proteomics identified changes in the expression of proteins involved in alcohol, lipid, and amino acid metabolism after ethanol feeding. At 1 and 3 months, 12 and 15 different proteins were differentially expressed. Of the identified proteins, down regulation of alcohol dehydrogenase (− 1.6) at 1 month and up regulation of aldehyde dehydrogenase (2.1) at 3 months could be a protective/adaptive mechanism against ethanol toxicity. In addition, betaine-homocysteine S-methyltransferase 2 a protein responsible for methionine metabolism and previously implicated in fatty liver development was significantly up regulated (1.4) at ethanol-induced fatty liver stage (1 month) while peroxiredoxin-1 was down regulated (− 1.5) at late fatty liver stage (3 months). Nonparametric analysis of the protein spots yielded fewer proteins and narrowed the list of possible markers and identified D-dopachrome tautomerase (− 1.7, at 3 months) as a possible marker for ethanol-induced early steatohepatitis. The observed differential regulation of proteins have potential to serve as biomarker signature for the detection of steatosis and its progression to steatohepatitis once validated in plasma/serum. -- Graphical abstract: The figure shows the Hierarchial cluster analysis of differentially expressed protein spots obtained after ethanol feeding for 1 (1–3

Extracellular histones disarrange vasoactive mediators release through a COX-NOS interaction in human endothelial cells.

Science.gov (United States)

Pérez-Cremades, Daniel; Bueno-Betí, Carlos; García-Giménez, José Luis; Ibañez-Cabellos, José Santiago; Hermenegildo, Carlos; Pallardó, Federico V; Novella, Susana

2017-08-01

Extracellular histones are mediators of inflammation, tissue injury and organ dysfunction. Interactions between circulating histones and vascular endothelial cells are key events in histone-mediated pathologies. Our aim was to investigate the implication of extracellular histones in the production of the major vasoactive compounds released by human endothelial cells (HUVECs), prostanoids and nitric oxide (NO). HUVEC exposed to increasing concentrations of histones (0.001 to 100 μg/ml) for 4 hrs induced prostacyclin (PGI2) production in a dose-dependent manner and decreased thromboxane A2 (TXA2) release at 100 μg/ml. Extracellular histones raised cyclooxygenase-2 (COX-2) and prostacyclin synthase (PGIS) mRNA and protein expression, decreased COX-1 mRNA levels and did not change thromboxane A2 synthase (TXAS) expression. Moreover, extracellular histones decreased both, eNOS expression and NO production in HUVEC. The impaired NO production was related to COX-2 activity and superoxide production since was reversed after celecoxib (10 μmol/l) and tempol (100 μmol/l) treatments, respectively. In conclusion, our findings suggest that extracellular histones stimulate the release of endothelial-dependent mediators through an up-regulation in COX-2-PGIS-PGI2 pathway which involves a COX-2-dependent superoxide production that decreases the activity of eNOS and the NO production. These effects may contribute to the endothelial cell dysfunction observed in histone-mediated pathologies. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Tissue-based map of the human proteome

DEFF Research Database (Denmark)

Uhlén, Mathias; Fagerberg, Linn; Hallström, Björn M.

2015-01-01

Resolving the molecular details of proteome variation in the different tissues and organs of the human body will greatly increase our knowledge of human biology and disease. Here, we present a map of the human tissue proteome based on an integrated omics approach that involves quantitative transc...

Involvement of extracellular matrix constituents in breast cancer

Energy Technology Data Exchange (ETDEWEB)

Lochter, Andre; Bissell, Mina J

1995-06-01

It has recently been established that the extracellular matrix is required for normal functional differentiation of mammary epithelia not only in culture, but also in vivo. The mechanisms by which extracellular matrix affects differentiation, as well as the nature of extracellular matrix constituents which have major impacts on mammary gland function, have only now begun to be dissected. The intricate variety of extracellular matrix-mediated events and the remarkable degree of plasticity of extracellular matrix structure and composition at virtually all times during ontogeny, make such studies difficult. Similarly, during carcinogenesis, the extracellular matrix undergoes gross alterations, the consequences of which are not yet precisely understood. Nevertheless, an increasing amount of data suggests that the extracellular matrix and extracellular matrix-receptors might participate in the control of most, if not all, of the successive stages of breast tumors, from appearance to progression and metastasis.

Plant redox proteomics

DEFF Research Database (Denmark)

Navrot, Nicolas; Finnie, Christine; Svensson, Birte

2011-01-01

PTMs in regulating enzymatic activities and controlling biological processes in plants. Notably, proteins controlling the cellular redox state, e.g. thioredoxin and glutaredoxin, appear to play dual roles to maintain oxidative stress resistance and regulate signal transduction pathways via redox PTMs......In common with other aerobic organisms, plants are exposed to reactive oxygen species resulting in formation of post-translational modifications related to protein oxidoreduction (redox PTMs) that may inflict oxidative protein damage. Accumulating evidence also underscores the importance of redox....... To get a comprehensive overview of these types of redox-regulated pathways there is therefore an emerging interest to monitor changes in redox PTMs on a proteome scale. Compared to some other PTMs, e.g. protein phosphorylation, redox PTMs have received less attention in plant proteome analysis, possibly...

Extracellular DNA metabolism in Haloferax volcanii

Directory of Open Access Journals (Sweden)

Scott eChimileski

2014-02-01

Full Text Available Extracellular DNA is found in all environments and is a dynamic component of the micro-bial ecosystem. Microbial cells produce and interact with extracellular DNA through many endogenous mechanisms. Extracellular DNA is processed and internalized for use as genetic information and as a major source of macronutrients, and plays several key roles within prokaryotic biofilms. Hypersaline sites contain some of the highest extracellular DNA con-centrations measured in nature–a potential rich source of carbon, nitrogen and phosphorus for halophilic microorganisms. We conducted DNA growth studies for the halophilic archaeon Haloferax volcanii DS2 and show that this model Halobacteriales strain is capable of using exogenous double-stranded DNA as a nutrient. Further experiments with varying medium composition, DNA concentration and DNA types revealed that DNA is utilized primarily as a phosphorus source, that growth on DNA is concentration-dependent and that DNA isolated from different sources is metabolized selectively, with a bias against highly divergent methylated DNA sources. Additionally, fluorescence microscopy experiments showed that labeled DNA colocalized with Haloferax volcanii cells. The gene Hvo_1477 was also identified using a comparative genomic approach as a factor likely to be involved in extracellular DNA processing at the cell surface, and deletion of Hvo_1477 created an H. volcanii strain deficient in its ability to grow on extracellular DNA. Widespread distribution of Hvo_1477 homologs in archaea suggests metabolism of extracellular DNA may be of broad ecological and physiological relevance in this domain of life.

Dermal extracellular lipid in birds.

Science.gov (United States)

Stromberg, M W; Hinsman, E J; Hullinger, R L

1990-01-01

A light and electron microscopic study of the skin of domestic chickens, seagulls, and antarctic penguins revealed abundant extracellular dermal lipid and intracellular epidermal lipid. Dermal lipid appeared ultrastructurally as extracellular droplets varying from less than 1 micron to more than 25 microns in diameter. The droplets were often irregularly contoured, sometimes round, and of relatively low electron density. Processes of fibrocytes were often seen in contact with extracellular lipid droplets. Sometimes a portion of such a droplet was missing, and this missing part appeared to have been "digested away" by the cell process. In places where cells or cell processes are in contact with fact droplets, there are sometimes extracellular membranous whorls or fragments which have been associated with the presence of fatty acids. Occasionally (in the comb) free fat particles were seen in intimate contact with extravasated erythrocytes. Fat droplets were seen in the lumen of small dermal blood and lymph vessels. We suggest that the dermal extracellular lipid originates in the adipocyte layer and following hydrolysis the free fatty acids diffuse into the epidermis. Here they become the raw material for forming the abundant neutral lipid contained in many of the epidermal cells of both birds and dolphins. The heretofore unreported presence and apparently normal utilization of abundant extracellular lipid in birds, as well as the presence of relatively large droplets of neutral lipid in dermal vessels, pose questions which require a thorough reappraisal of present concepts of the ways in which fat is distributed and utilized in the body.

Membrane proteomics of phagosomes suggests a connection to autophagy

Energy Technology Data Exchange (ETDEWEB)

Shui, Wenqing; Sheu, Leslie; Liu, Jun; Smart, Brian; Petzold, Christopher J.; Hsieh, Tsung-yen; Pitcher, Austin; Keasling*, Jay D.; Bertozzi*, Carolyn R.

2008-11-25

Phagocytosis is the central process by which macrophage cellsinternalize and eliminate infectious microbes as well as apoptoticcells. During maturation, phagosomes containing engulfed particlesfuse with various endosomal compartments through theaction of regulatory molecules on the phagosomal membrane. Inthis study, we performed a proteomic analysis of the membranefraction from latex bead-containing (LBC) phagosomes isolatedfrom macrophages. The profile, which comprised 546 proteins,suggests diverse functions of the phagosome and potential connectionsto secretory processes, toll-like receptor signaling, andautophagy. Many identified proteins were not previously knownto reside in the phagosome. We characterized several proteins inLBC phagosomes that change in abundance on induction of autophagy,a process that has been previously implicated in the hostdefense against microbial pathogens. These observations suggestcrosstalk between autophagy and phagocytosis that may be relevantto the innate immune response of macrophages.

PatternLab for proteomics: a tool for differential shotgun proteomics

Directory of Open Access Journals (Sweden)

Yates John R

2008-07-01

Full Text Available Abstract Background A goal of proteomics is to distinguish between states of a biological system by identifying protein expression differences. Liu et al. demonstrated a method to perform semi-relative protein quantitation in shotgun proteomics data by correlating the number of tandem mass spectra obtained for each protein, or "spectral count", with its abundance in a mixture; however, two issues have remained open: how to normalize spectral counting data and how to efficiently pinpoint differences between profiles. Moreover, Chen et al. recently showed how to increase the number of identified proteins in shotgun proteomics by analyzing samples with different MS-compatible detergents while performing proteolytic digestion. The latter introduced new challenges as seen from the data analysis perspective, since replicate readings are not acquired. Results To address the open issues above, we present a program termed PatternLab for proteomics. This program implements existing strategies and adds two new methods to pinpoint differences in protein profiles. The first method, ACFold, addresses experiments with less than three replicates from each state or having assays acquired by different protocols as described by Chen et al. ACFold uses a combined criterion based on expression fold changes, the AC test, and the false-discovery rate, and can supply a "bird's-eye view" of differentially expressed proteins. The other method addresses experimental designs having multiple readings from each state and is referred to as nSVM (natural support vector machine because of its roots in evolutionary computing and in statistical learning theory. Our observations suggest that nSVM's niche comprises projects that select a minimum set of proteins for classification purposes; for example, the development of an early detection kit for a given pathology. We demonstrate the effectiveness of each method on experimental data and confront them with existing strategies

Comparative proteomics of cerebrospinal fluid reveals a predictive model for differential diagnosis of pneumococcal, meningococcal, and enteroviral meningitis, and novel putative therapeutic targets

Science.gov (United States)

2015-01-01

Background Meningitis is the inflammation of the meninges in response to infection or chemical agents. While aseptic meningitis, most frequently caused by enteroviruses, is usually benign with a self-limiting course, bacterial meningitis remains associated with high morbidity and mortality rates, despite advances in antimicrobial therapy and intensive care. Fast and accurate differential diagnosis is crucial for assertive choice of the appropriate therapeutic approach for each form of meningitis. Methods We used 2D-PAGE and mass spectrometry to identify the cerebrospinal fluid proteome specifically related to the host response to pneumococcal, meningococcal, and enteroviral meningitis. The disease-specific proteome signatures were inspected by pathway analysis. Results Unique cerebrospinal fluid proteome signatures were found to the three aetiological forms of meningitis investigated, and a qualitative predictive model with four protein markers was developed for the differential diagnosis of these diseases. Nevertheless, pathway analysis of the disease-specific proteomes unveiled that Kallikrein-kinin system may play a crucial role in the pathophysiological mechanisms leading to brain damage in bacterial meningitis. Proteins taking part in this cellular process are proposed as putative targets to novel adjunctive therapies. Conclusions Comparative proteomics of cerebrospinal fluid disclosed candidate biomarkers, which were combined in a qualitative and sequential predictive model with potential to improve the differential diagnosis of pneumococcal, meningococcal and enteroviral meningitis. Moreover, we present the first evidence of the possible implication of Kallikrein-kinin system in the pathophysiology of bacterial meningitis. PMID:26040285

Proteomic Insights on the Metabolism of Penicillium janczewskii during the Biotransformation of the Plant Terpenoid Labdanolic Acid

Directory of Open Access Journals (Sweden)

Isabel Martins

2017-07-01

Full Text Available Plant terpenoids compose a natural source of chemodiversity of exceptional value. Many of these compounds own biological/pharmacological activity, others are regarded as unique chemical skeletons for the synthesis of derivatives with improved properties. Functional chemical modification of terpenoids through biotransformation frequently relies on the use of Ascomycota strains, but information on major cellular responses is still largely lacking. Penicillium janczewskii mediates a stereo-selective hydroxylation of labdanolic acid (LA—terpenoid found abundantly in Cistus ladanifer—producing 3β-hydroxy-labdanolic acid with yields >90%. Herein, combined analyses of mycelial and extracellular differential proteomes demonstrated that the plant terpenoid increased stress responses, especially against oxidative stress (e.g., accumulation of superoxide dismutase and apparently altered mitochondria functioning. One putative cytochrome P450 monooxygenase differentially accumulated in the secretome and the terpenoid bioconversion was inhibited in vivo in the presence of a P450 inhibitor. The stereo-selective hydroxylation of the plant terpenoid is likely mediated by P450 enzymes, yet its unequivocal identity remains unclear. To the best of our knowledge, this is the first time that proteomics was used to investigate how a plant terpenoid impacts the metabolism of a filamentous fungus during its efficiently biotransformation. Our findings may encourage the development of new strategies for the valorization of plant natural resources through biotechnology.

Clinical proteomic analysis of scrub typhus infection.

Science.gov (United States)

Park, Edmond Changkyun; Lee, Sang-Yeop; Yun, Sung Ho; Choi, Chi-Won; Lee, Hayoung; Song, Hyun Seok; Jun, Sangmi; Kim, Gun-Hwa; Lee, Chang-Seop; Kim, Seung Il

2018-01-01

Scrub typhus is an acute and febrile infectious disease caused by the Gram-negative α-proteobacterium Orientia tsutsugamushi from the family Rickettsiaceae that is widely distributed in Northern, Southern and Eastern Asia. In the present study, we analysed the serum proteome of scrub typhus patients to investigate specific clinical protein patterns in an attempt to explain pathophysiology and discover potential biomarkers of infection. Serum samples were collected from three patients (before and after treatment with antibiotics) and three healthy subjects. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by liquid chromatography-tandem mass spectrometry was performed to identify differentially abundant proteins using quantitative proteomic approaches. Bioinformatic analysis was then performed using Ingenuity Pathway Analysis. Proteomic analysis identified 236 serum proteins, of which 32 were differentially expressed in normal subjects, naive scrub typhus patients and patients treated with antibiotics. Comparative bioinformatic analysis of the identified proteins revealed up-regulation of proteins involved in immune responses, especially complement system, following infection with O. tsutsugamushi , and normal expression was largely rescued by antibiotic treatment. This is the first proteomic study of clinical serum samples from scrub typhus patients. Proteomic analysis identified changes in protein expression upon infection with O. tsutsugamushi and following antibiotic treatment. Our results provide valuable information for further investigation of scrub typhus therapy and diagnosis.

The path to enlightenment: making sense of genomic and proteomic information.

Science.gov (United States)

Maurer, Martin H

2004-05-01

Whereas genomics describes the study of genome, mainly represented by its gene expression on the DNA or RNA level, the term proteomics denotes the study of the proteome, which is the protein complement encoded by the genome. In recent years, the number of proteomic experiments increased tremendously. While all fields of proteomics have made major technological advances, the biggest step was seen in bioinformatics. Biological information management relies on sequence and structure databases and powerful software tools to translate experimental results into meaningful biological hypotheses and answers. In this resource article, I provide a collection of databases and software available on the Internet that are useful to interpret genomic and proteomic data. The article is a toolbox for researchers who have genomic or proteomic datasets and need to put their findings into a biological context.

Proteomic interrogation of human chromatin.

Directory of Open Access Journals (Sweden)

Mariana P Torrente

Full Text Available Chromatin proteins provide a scaffold for DNA packaging and a basis for epigenetic regulation and genomic maintenance. Despite understanding its functional roles, mapping the chromatin proteome (i.e. the "Chromatome" is still a continuing process. Here, we assess the biological specificity and proteomic extent of three distinct chromatin preparations by identifying proteins in selected chromatin-enriched fractions using mass spectrometry-based proteomics. These experiments allowed us to produce a chromatin catalog, including several proteins ranging from highly abundant histone proteins to less abundant members of different chromatin machinery complexes. Using a Normalized Spectral Abundance Factor approach, we quantified relative abundances of the proteins across the chromatin enriched fractions giving a glimpse into their chromosomal abundance. The large-scale data sets also allowed for the discovery of a variety of novel post-translational modifications on the identified chromatin proteins. With these comparisons, we find one of the probed methods to be qualitatively superior in specificity for chromatin proteins, but inferior in proteomic extent, evidencing a compromise that must be made between biological specificity and broadness of characterization. Additionally, we attempt to identify proteins in eu- and heterochromatin, verifying the enrichments by characterizing the post-translational modifications detected on histone proteins from these chromatin regions. In summary, our results provide insights into the value of different methods to extract chromatin-associated proteins and provide starting points to study the factors that may be involved in directing gene expression and other chromatin-related processes.

Biofilm-specific extracellular matrix proteins of non-typeable Haemophilus influenzae

Science.gov (United States)

Wu, Siva; Baum, Marc M.; Kerwin, James; Guerrero-Given, Debbie; Webster, Simon; Schaudinn, Christoph; VanderVelde, David; Webster, Paul

2014-01-01

Non-typeable Haemophilus influenzae (NTHi), a human respiratory tract pathogen can form colony biofilms in vitro. Bacterial cells and the amorphous extracellular matrix (ECM) constituting the biofilm can be separated using sonication. The ECM from 24 hr and 96 hr NTHi biofilms contained polysaccharides and proteinaceous components as detected by NMR and FTIR spectroscopy. More conventional chemical assays on the biofilm ECM confirmed the presence of these components and also DNA. Proteomics revealed eighteen proteins present in biofilm ECM that were not detected in planktonic bacteria. One ECM protein was unique to 24 hr biofilms, two were found only in 96 hr biofilms, and fifteen were present in the ECM of both 24 hr and 96 hr NTHi biofilms. All proteins identified were either associated with bacterial membranes or were cytoplasmic proteins. Immunocytochemistry showed two of the identified proteins, a DNA-directed RNA polymerase and the outer membrane protein OMP P2, associated with bacteria and biofilm ECM. Identification of biofilm-specific proteins present in immature biofilms is an important step in understanding the in vitro process of NTHi biofilm formation. The presence of a cytoplasmic protein and a membrane protein in the biofilm ECM of immature NTHi biofilms suggests that bacterial cell lysis may be a feature of early biofilm formation. PMID:24942343

Extracellular matrix assembly in extreme acidic eukaryotic biofilms and their possible implications in heavy metal adsorption

International Nuclear Information System (INIS)

Aguilera, Angeles; Souza-Egipsy, Virginia; San Martin-Uriz, Patxi; Amils, Ricardo

2008-01-01

To evaluate the importance of the extracellular matrix in relation to heavy metal binding capacity in extreme acidic environments, the extracellular polymeric substances (EPS) composition of 12 biofilms isolated from Rio Tinto (SW, Spain) was analyzed. Each biofilm was composed mainly by one or two species of eukaryotes, although other microorganisms were present. EPS ranged from 130 to 439 mg g -1 biofilm dry weight, representing between 15% and the 40% of the total biofilm dry weight (DW). Statistically significant differences (p -1 dry weight; 10% to 30% of the total biofilm dry weight. Capsular EPS ranged from 50 to 318 mg g -1 dry weight; 5% to 30% of the total biofilm dry weight. Seven of the 12 biofilms showed higher amounts of capsular than colloidal EPS (p -1 biofilm dry weight, reaching up to 16% of the total composition. In general, the heavy metal composition of the EPS extracted from the biofilms closely resembled the metal composition of the water from which the biofilms were collected

Paving a Path to Understanding Metabolic Responses to Iron Bioavailability: Global Proteomic Analysis of Crocosphaera watsonii

Science.gov (United States)

Gauglitz, J.; McIlvin, M. R.; Moran, D. M.; Waterbury, J. B.; Saito, M. A.

2016-02-01

Marine diazotrophic cyanobacteria provide a key source of new nitrogen into the oceans and are important contributors to primary production. The geographic distribution of these cyanobacteria is impacted by available iron and phosphorus as well as environmental conditions such as temperature, however available iron concentrations are thought to be particularly critical due to the high demand for iron in cellular processes. Iron bioavailability and microorganismal adaptations to low iron environments may thus play a key role in dictating community structure, however the mechanisms by which cyanobacteria acquire iron and regulate its uptake are not well defined. In this study, the unicellular diazotroph, Crocosphaera watsonii WH8501, was acclimated to a range of bioavailable iron concentrations (from 0.001nM to 8.13nM Fe') using trace metal clean culturing techniques and the proteomes were analyzed by LC/MS-MS. Physiological and proteomic data indicate three distinct phenotypic ranges: iron-replete, iron-limited, and iron-starved. Trends in photosynthetic, carbon fixation and iron storage proteins across the iron gradient indicate that the C. watsonii proteome responds directly to iron availability. Further analysis of relative protein expression, which describes the physiological state of the cell, will lead to insights into how C. watsonii is able to adapt to iron-limited conditions and the resulting biogeochemical implications will be discussed.

The state of proteome profiling in the fungal genus Aspergillus.

Science.gov (United States)

Kim, Yonghyun; Nandakumar, M P; Marten, Mark R

2008-03-01

Aspergilli are an important genus of filamentous fungi that contribute to a multibillion dollar industry. Since many fungal genome sequencing were recently completed, it would be advantageous to profile their proteome to better understand the fungal cell factory. Here, we review proteomic data generated for the Aspergilli in recent years. Thus far, a combined total of 28 cell surface, 102 secreted and 139 intracellular proteins have been identified based on 10 different studies on Aspergillus proteomics. A summary proteome map highlighting identified proteins in major metabolic pathway is presented.

Comparative proteomic analysis of human malignant ascitic fluids for the development of gastric cancer biomarkers.

Science.gov (United States)

Jin, Jonghwa; Son, Minsoo; Kim, Hyeyoon; Kim, Hyeyeon; Kong, Seong-Ho; Kim, Hark Kyun; Kim, Youngsoo; Han, Dohyun

2018-04-11

Malignant ascites is a sign of peritoneal seeding, which is one of the most frequent forms of incurable distant metastasis. Because the development of malignant ascites is associated with an extremely poor prognosis, determining whether it resulted from peritoneal seeding has critical clinical implications in diagnosis, choice of treatment, and active surveillance. At present, the molecular characterizations of malignant ascites are especially limited in case of gastric cancer. We aimed to identify malignant ascites-specific proteins that may contribute to the development of alternative methods for diagnosis and therapeutic monitoring and also increase our understanding of the pathophysiology of peritoneal seeding. First, comprehensive proteomic strategies were employed to construct an in-depth proteome of ascitic fluids. Label-free quantitative proteomic analysis was subsequently performed to identify candidates that can differentiate between malignant ascitic fluilds of gastric cancer patients from benign ascitic fluids. Finally, two candidate proteins were verified by ELISA in 84 samples with gastric cancer or liver cirrhosis. Comprehensive proteome profiling resulted in the identification of 5347 ascites proteins. Using label-free quantification, we identified 299 proteins that were differentially expressed in ascitic fluids between liver cirrhosis and stage IV gastric cancer patients. In addition, we identified 645 proteins that were significantly expressed in ascitic fluids between liver cirrhosis and gastric cancer patients with peritoneal seeding. Finally, Gastriscin and Periostin that can distinguish malignant ascites from benign ascites were verified by ELISA. This study identified and verified protein markers that can distinguish malignant ascites with or without peritoneal seeding from benign ascites. Consequently, our results could be a significant resource for gastric cancer research and biomarker discovery in the diagnosis of malignant ascites

Proteome map of Aspergillus nidulans during osmoadaptation.

Science.gov (United States)

Kim, Yonghyun; Nandakumar, M P; Marten, Mark R

2007-09-01

The model filamentous fungus Aspergillus nidulans, when grown in a moderate level of osmolyte (+0.6M KCl), was previously found to have a significantly reduced cell wall elasticity (Biotech Prog, 21:292, 2005). In this study, comparative proteomic analysis via two-dimensional gel electrophoresis (2de) and matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry was used to assess molecular level events associated with this phenomenon. Thirty of 90 differentially expressed proteins were identified. Sequence homology and conserved domains were used to assign probable function to twenty-one proteins currently annotated as "hypothetical." In osmoadapted cells, there was an increased expression of glyceraldehyde-3-phosphate dehydrogenase and aldehyde dehydrogenase, as well as a decreased expression of enolase, suggesting an increased glycerol biosynthesis and decreased use of the TCA cycle. There also was an increased expression of heat shock proteins and Shp1-like protein degradation protein, implicating increased protein turnover. Five novel osmoadaptation proteins of unknown functions were also identified.

Scientific Workflow Management in Proteomics

Science.gov (United States)

de Bruin, Jeroen S.; Deelder, André M.; Palmblad, Magnus

2012-01-01

Data processing in proteomics can be a challenging endeavor, requiring extensive knowledge of many different software packages, all with different algorithms, data format requirements, and user interfaces. In this article we describe the integration of a number of existing programs and tools in Taverna Workbench, a scientific workflow manager currently being developed in the bioinformatics community. We demonstrate how a workflow manager provides a single, visually clear and intuitive interface to complex data analysis tasks in proteomics, from raw mass spectrometry data to protein identifications and beyond. PMID:22411703

PROTEOMICS in aquaculture

DEFF Research Database (Denmark)

Rodrigues, Pedro M.; Silva, Tomé S.; Dias, Jorge

2012-01-01

Over the last forty years global aquaculture presented a growth rate of 6.9% per annum with an amazing production of 52.5million tonnes in 2008, and a contribution of 43% of aquatic animal food for human consumption. In order to meet the world's health requirements of fish protein, a continuous...... growth in production is still expected for decades to come. Aquaculture is, though, a very competitive market, and a global awareness regarding the use of scientific knowledge and emerging technologies to obtain a better farmed organism through a sustainable production has enhanced the importance...... questions and the role of proteomics in their investigation, outlining the advantages, disadvantages and future challenges. A brief description of the proteomics technical approaches will be presented. Special focus will be on the latest trends related to the aquaculture production of fish with defined...

Implementation of proteomics for cancer research: past, present, and future.

Science.gov (United States)

Karimi, Parisa; Shahrokni, Armin; Ranjbar, Mohammad R Nezami

2014-01-01

Cancer is the leading cause of the death, accounts for about 13% of all annual deaths worldwide. Many different fields of science are collaborating together studying cancer to improve our knowledge of this lethal disease, and find better solutions for diagnosis and treatment. Proteomics is one of the most recent and rapidly growing areas in molecular biology that helps understanding cancer from an omics data analysis point of view. The human proteome project was officially initiated in 2008. Proteomics enables the scientists to interrogate a variety of biospecimens for their protein contents and measure the concentrations of these proteins. Current necessary equipment and technologies for cancer proteomics are mass spectrometry, protein microarrays, nanotechnology and bioinformatics. In this paper, we provide a brief review on proteomics and its application in cancer research. After a brief introduction including its definition, we summarize the history of major previous work conducted by researchers, followed by an overview on the role of proteomics in cancer studies. We also provide a list of different utilities in cancer proteomics and investigate their advantages and shortcomings from theoretical and practical angles. Finally, we explore some of the main challenges and conclude the paper with future directions in this field.

Quantitative N-linked Glycoproteomics of Myocardial Ischemia and Reperfusion Injury Reveals Early Remodeling in the Extracellular Environment

DEFF Research Database (Denmark)

Parker, Benjamin L; Palmisano, Giuseppe; Edwards, Alistair V G

2011-01-01

, while dimethyl labeling confirmed 46 of these and revealed an additional 62 significant changes. These were mainly from predicted extracellular matrix and basement membrane proteins that are implicated in cardiac remodeling. Analysis of N-glycans released from myocardial proteins suggest...... that the observed changes were not due to significant alterations in N-glycan structures. Altered proteins included the collagen-laminin-integrin complexes and collagen assembly enzymes, cadherins, mast cell proteases, proliferation-associated secreted protein acidic and rich in cysteine, and microfibril......Extracellular and cell surface proteins are generally modified with N-linked glycans and glycopeptide enrichment is an attractive tool to analyze these proteins. The role of N-linked glycoproteins in cardiovascular disease, particularly ischemia and reperfusion injury, is poorly understood...

KLK6 proteolysis is implicated in the turnover and uptake of extracellular alpha-synuclein species.

Science.gov (United States)

Pampalakis, Georgios; Sykioti, Vasia-Samantha; Ximerakis, Methodios; Stefanakou-Kalakou, Ioanna; Melki, Ronald; Vekrellis, Kostas; Sotiropoulou, Georgia

2017-02-28

KLK6 is a serine protease highly expressed in the nervous system. In synucleinopathies, including Parkinson disease, the levels of KLK6 inversely correlate with α-synuclein in CSF. Recently, we suggested that recombinant KLK6 mediates the degradation of extracellular α-synuclein directly and via a proteolytic cascade that involves unidentified metalloproteinase(s). Here, we show that recombinant and naturally secreted KLK6 can readily cleave α-synuclein fibrils that have the potential for cell-to-cell propagation in "a prion-like mechanism". Importantly, KLK6-deficient primary cortical neurons have increased ability for α-synuclein fibril uptake. We also demonstrate that KLK6 activates proMMP2, which in turn can cleave α-synuclein. The repertoire of proteases activated by KLK6 in a neuronal environment was analyzed by degradomic profiling, which also identified ADAMTS19 and showed that KLK6 has a limited number of substrates indicating specific biological functions such as the regulation of α-synuclein turnover. We generated adenoviral vectors for KLK6 delivery and demonstrated that the levels of extracellular α-synuclein can be reduced by neuronally secreted KLK6. Our findings open the possibility to exploit KLK6 as a novel therapeutic target for Parkinson disease and other synucleinopathies.

Positional proteomics in the era of the human proteome project on the doorstep of precision medicine.

Science.gov (United States)

Eckhard, Ulrich; Marino, Giada; Butler, Georgina S; Overall, Christopher M

2016-03-01

Proteolytic processing is a pervasive and irreversible post-translational modification that expands the protein universe by generating new proteoforms (protein isoforms). Unlike signal peptide or prodomain removal, protease-generated proteoforms can rarely be predicted from gene sequences. Positional proteomic techniques that enrich for N- or C-terminal peptides from proteomes are indispensable for a comprehensive understanding of a protein's function in biological environments since protease cleavage frequently results in altered protein activity and localization. Proteases often process other proteases and protease inhibitors which perturbs proteolytic networks and potentiates the initial cleavage event to affect other molecular networks and cellular processes in physiological and pathological conditions. This review is aimed at researchers with a keen interest in state of the art systems level positional proteomic approaches that: (i) enable the study of complex protease-protease, protease-inhibitor and protease-substrate crosstalk and networks; (ii) allow the identification of proteolytic signatures as candidate disease biomarkers; and (iii) are expected to fill the Human Proteome Project missing proteins gap. We predict that these methodologies will be an integral part of emerging precision medicine initiatives that aim to customize healthcare, converting reactive medicine into a personalized and proactive approach, improving clinical care and maximizing patient health and wellbeing, while decreasing health costs by eliminating ineffective therapies, trial-and-error prescribing, and adverse drug effects. Such initiatives require quantitative and functional proteome profiling and dynamic disease biomarkers in addition to current pharmacogenomics approaches. With proteases at the pathogenic center of many diseases, high-throughput protein termini identification techniques such as TAILS (Terminal Amine Isotopic Labeling of Substrates) and COFRADIC (COmbined

Effects of sleep deprivation on extracellular serotonin in hippocampus and frontal cortex of the rat

OpenAIRE

Bjorvatn, B; Grønli, J; Hamre, F; Sørensen, E; Fiske, E; Bjorkum, Alvhild Alette; Portas, CM; Ursin, R

2002-01-01

Sleep deprivation improves the mood of depressed patients, but the exact mechanism behind this effect is unclear. An enhancement of serotonergic neurotransmission has been suggested. In this study, we used in vivo microdialysis to monitor extracellular serotonin in the hippocampus and the frontal cortex of rats during an 8 h sleep deprivation period. These brain regions were selected since both have been implicated in depression. The behavioral state of the animal was continuously monitored b...

The Proteomics Big Challenge for Biomarkers and New Drug-Targets Discovery

Science.gov (United States)

Savino, Rocco; Paduano, Sergio; Preianò, Mariaimmacolata; Terracciano, Rosa

2012-01-01

In the modern process of drug discovery, clinical, functional and chemical proteomics can converge and integrate synergies. Functional proteomics explores and elucidates the components of pathways and their interactions which, when deregulated, lead to a disease condition. This knowledge allows the design of strategies to target multiple pathways with combinations of pathway-specific drugs, which might increase chances of success and reduce the occurrence of drug resistance. Chemical proteomics, by analyzing the drug interactome, strongly contributes to accelerate the process of new druggable targets discovery. In the research area of clinical proteomics, proteome and peptidome mass spectrometry-profiling of human bodily fluid (plasma, serum, urine and so on), as well as of tissue and of cells, represents a promising tool for novel biomarker and eventually new druggable targets discovery. In the present review we provide a survey of current strategies of functional, chemical and clinical proteomics. Major issues will be presented for proteomic technologies used for the discovery of biomarkers for early disease diagnosis and identification of new drug targets. PMID:23203042

Single muscle fiber proteomics reveals unexpected mitochondrial specialization

DEFF Research Database (Denmark)

Murgia, Marta; Nagaraj, Nagarjuna; Deshmukh, Atul S

2015-01-01

and unbiased proteomics methods yielded the same subtype assignment. We discovered novel subtype-specific features, most prominently mitochondrial specialization of fiber types in substrate utilization. The fiber type-resolved proteomes can be applied to a variety of physiological and pathological conditions...

Shaping Biological Knowledge: Applications in Proteomics

Directory of Open Access Journals (Sweden)

R. Appel

2006-04-01

Full Text Available The central dogma of molecular biology has provided a meaningful principle for data integration in the field of genomics. In this context, integration reflects the known transitions from a chromosome to a protein sequence: transcription, intron splicing, exon assembly and translation. There is no such clear principle for integrating proteomics data, since the laws governing protein folding and interactivity are not quite understood. In our effort to bring together independent pieces of information relative to proteins in a biologically meaningful way, we assess the bias of bioinformatics resources and consequent approximations in the framework of small-scale studies. We analyse proteomics data while following both a data-driven (focus on proteins smaller than 10 kDa and a hypothesis-driven (focus on whole bacterial proteomes approach. These applications are potentially the source of specialized complements to classical biological ontologies.

Shaping biological knowledge: applications in proteomics.

Science.gov (United States)

Lisacek, F; Chichester, C; Gonnet, P; Jaillet, O; Kappus, S; Nikitin, F; Roland, P; Rossier, G; Truong, L; Appel, R

2004-01-01

The central dogma of molecular biology has provided a meaningful principle for data integration in the field of genomics. In this context, integration reflects the known transitions from a chromosome to a protein sequence: transcription, intron splicing, exon assembly and translation. There is no such clear principle for integrating proteomics data, since the laws governing protein folding and interactivity are not quite understood. In our effort to bring together independent pieces of information relative to proteins in a biologically meaningful way, we assess the bias of bioinformatics resources and consequent approximations in the framework of small-scale studies. We analyse proteomics data while following both a data-driven (focus on proteins smaller than 10 kDa) and a hypothesis-driven (focus on whole bacterial proteomes) approach. These applications are potentially the source of specialized complements to classical biological ontologies.

The Extracellular Matrix of Candida albicans Biofilms Impairs Formation of Neutrophil Extracellular Traps.

Science.gov (United States)

Johnson, Chad J; Cabezas-Olcoz, Jonathan; Kernien, John F; Wang, Steven X; Beebe, David J; Huttenlocher, Anna; Ansari, Hamayail; Nett, Jeniel E

2016-09-01

Neutrophils release extracellular traps (NETs) in response to planktonic C. albicans. These complexes composed of DNA, histones, and proteins inhibit Candida growth and dissemination. Considering the resilience of Candida biofilms to host defenses, we examined the neutrophil response to C. albicans during biofilm growth. In contrast to planktonic C. albicans, biofilms triggered negligible release of NETs. Time lapse imaging confirmed the impairment in NET release and revealed neutrophils adhering to hyphae and migrating on the biofilm. NET inhibition depended on an intact extracellular biofilm matrix as physical or genetic disruption of this component resulted in NET release. Biofilm inhibition of NETosis could not be overcome by protein kinase C activation via phorbol myristate acetate (PMA) and was associated with suppression of neutrophil reactive oxygen species (ROS) production. The degree of impaired NET release correlated with resistance to neutrophil attack. The clinical relevance of the role for extracellular matrix in diminishing NET production was corroborated in vivo using a rat catheter model. The C. albicans pmr1Δ/Δ, defective in production of matrix mannan, appeared to elicit a greater abundance of NETs by scanning electron microscopy imaging, which correlated with a decreased fungal burden. Together, these findings show that C. albicans biofilms impair neutrophil response through an inhibitory pathway induced by the extracellular matrix.

The Path to Enlightenment: Making Sense of Genomic and Proteomic Information

OpenAIRE

Maurer, Martin H.

2016-01-01

Whereas genomics describes the study of genome, mainly represented by its gene expression on the DNA or RNA level, the term proteomics denotes the study of the proteome, which is the protein complement encoded by the genome. In recent years, the number of proteomic experiments increased tremendously. While all fields of proteomics have made major technological advances, the biggest step was seen in bioinformatics. Biological information management relies on sequence and structure databases an...

Proteomic analysis of blastema formation in regenerating axolotl limbs

Directory of Open Access Journals (Sweden)

Nye Holly LD

2009-11-01

Full Text Available Abstract Background Following amputation, urodele salamander limbs reprogram somatic cells to form a blastema that self-organizes into the missing limb parts to restore the structure and function of the limb. To help understand the molecular basis of blastema formation, we used quantitative label-free liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS-based methods to analyze changes in the proteome that occurred 1, 4 and 7 days post amputation (dpa through the mid-tibia/fibula of axolotl hind limbs. Results We identified 309 unique proteins with significant fold change relative to controls (0 dpa, representing 10 biological process categories: (1 signaling, (2 Ca2+ binding and translocation, (3 transcription, (4 translation, (5 cytoskeleton, (6 extracellular matrix (ECM, (7 metabolism, (8 cell protection, (9 degradation, and (10 cell cycle. In all, 43 proteins exhibited exceptionally high fold changes. Of these, the ecotropic viral integrative factor 5 (EVI5, a cell cycle-related oncoprotein that prevents cells from entering the mitotic phase of the cell cycle prematurely, was of special interest because its fold change was exceptionally high throughout blastema formation. Conclusion Our data were consistent with previous studies indicating the importance of inositol triphosphate and Ca2+ signaling in initiating the ECM and cytoskeletal remodeling characteristic of histolysis and cell dedifferentiation. In addition, the data suggested that blastema formation requires several mechanisms to avoid apoptosis, including reduced metabolism, differential regulation of proapoptotic and antiapoptotic proteins, and initiation of an unfolded protein response (UPR. Since there is virtually no mitosis during blastema formation, we propose that high levels of EVI5 function to arrest dedifferentiated cells somewhere in the G1/S/G2 phases of the cell cycle until they have accumulated under the wound epidermis and enter mitosis in response to

Bayesian methods for proteomic biomarker development

Directory of Open Access Journals (Sweden)

Belinda Hernández

2015-12-01

In this review we provide an introduction to Bayesian inference and demonstrate some of the advantages of using a Bayesian framework. We summarize how Bayesian methods have been used previously in proteomics and other areas of bioinformatics. Finally, we describe some popular and emerging Bayesian models from the statistical literature and provide a worked tutorial including code snippets to show how these methods may be applied for the evaluation of proteomic biomarkers.

Proteomic analysis of mitochondria in respiratory epithelial cells infected with human respiratory syncytial virus and functional implications for virus and cell biology.

Science.gov (United States)

Munday, Diane C; Howell, Gareth; Barr, John N; Hiscox, Julian A

2015-03-01

The aim of this study was to quantitatively characterise the mitochondrial proteome of airway epithelial cells infected with human respiratory syncytial virus (HRSV), a major cause of paediatric illness. Quantitative proteomics, underpinned by stable isotope labelling with amino acids in cell culture, coupled to LC-MS/MS, was applied to mitochondrial fractions prepared from HRSV-infected and mock-infected cells 12 and 24 h post-infection. Datasets were analysed using ingenuity pathway analysis, and the results were validated and characterised using bioimaging, targeted inhibition and gene depletion. The data quantitatively indicated that antiviral signalling proteins converged on mitochondria during HRSV infection. The mitochondrial receptor protein Tom70 was found to act in an antiviral manner, while its chaperone, Hsp90, was confirmed to be a positive viral factor. Proteins associated with different organelles were also co-enriched in the mitochondrial fractions from HRSV-infected cells, suggesting that alterations in organelle dynamics and membrane associations occur during virus infection. Protein and pathway-specific alterations occur to the mitochondrial proteome in a spatial and temporal manner during HRSV infection, suggesting that this organelle may have altered functions. These could be targeted as part of potential therapeutic strategies to disrupt virus biology. © 2014 Royal Pharmaceutical Society.

Transcriptional and proteomic analysis of the Aspergillus fumigatus ΔprtT protease-deficient mutant.

Science.gov (United States)

Hagag, Shelly; Kubitschek-Barreira, Paula; Neves, Gabriela W P; Amar, David; Nierman, William; Shalit, Itamar; Shamir, Ron; Lopes-Bezerra, Leila; Osherov, Nir

2012-01-01

Aspergillus fumigatus is the most common opportunistic mold pathogen of humans, infecting immunocompromised patients. The fungus invades the lungs and other organs, causing severe damage. Penetration of the pulmonary epithelium is a key step in the infectious process. A. fumigatus produces extracellular proteases to degrade the host structural barriers. The A. fumigatus transcription factor PrtT controls the expression of multiple secreted proteases. PrtT shows similarity to the fungal Gal4-type Zn(2)-Cys(6) DNA-binding domain of several transcription factors. In this work, we further investigate the function of this transcription factor by performing a transcriptional and a proteomic analysis of the ΔprtT mutant. Unexpectedly, microarray analysis revealed that in addition to the expected decrease in protease expression, expression of genes involved in iron uptake and ergosterol synthesis was dramatically decreased in the ΔprtT mutant. A second finding of interest is that deletion of prtT resulted in the upregulation of four secondary metabolite clusters, including genes for the biosynthesis of toxic pseurotin A. Proteomic analysis identified reduced levels of three secreted proteases (ALP1 protease, TppA, AFUA_2G01250) and increased levels of three secreted polysaccharide-degrading enzymes in the ΔprtT mutant possibly in response to its inability to derive sufficient nourishment from protein breakdown. This report highlights the complexity of gene regulation by PrtT, and suggests a potential novel link between the regulation of protease secretion and the control of iron uptake, ergosterol biosynthesis and secondary metabolite production in A. fumigatus.

Extracellular matrix and growth factor engineering for controlled angiogenesis in regenerative medicine

Directory of Open Access Journals (Sweden)

Mikaël M Martino

2015-04-01

Full Text Available Blood vessel growth plays a key role in regenerative medicine, both to restore blood supply to ischemic tissues and to ensure rapid vascularization of clinical-size tissue-engineered grafts. For example, vascular endothelial growth factor (VEGF is the master regulator of physiological blood vessel growth and is one of the main molecular targets of therapeutic angiogenesis approaches. However, angiogenesis is a complex process and there is a need to develop rational therapeutic strategies based on a firm understanding of basic vascular biology principles, as evidenced by the disappointing results of initial clinical trials of angiogenic factor delivery. In particular, the spatial localization of angiogenic signals in the extracellular matrix is crucial to ensure the proper assembly and maturation of new vascular structures. Here we discuss the therapeutic implications of matrix interactions of angiogenic factors, with a special emphasis on VEGF, as well as provide an overview of current approaches, based on protein and biomaterial engineering that mimic the regulatory functions of extracellular matrix to optimize the signaling microenvironment of vascular growth factors.

Proteomics and circadian rhythms: It’s all about signaling!

Science.gov (United States)

Mauvoisin, Daniel; Dayon, Loïc; Gachon, Frédéric; Kussmann, Martin

2014-01-01

1. Abstract Proteomic technologies using mass spectrometry (MS) offer new perspectives in circadian biology, in particular the possibility to study posttranslational modifications (PTMs). To date, only very few studies have been carried out to decipher the rhythmicity of protein expression in mammals with large-scale proteomics. Although signaling has been shown to be of high relevance, comprehensive characterization studies of PTMs are even more rare. This review aims at describing the actual landscape of circadian proteomics and the opportunities and challenges appearing on the horizon. Emphasis was given to signaling processes for their role in metabolic heath as regulated by circadian clocks and environmental factors. Those signaling processes are expected to be better and more deeply characterized in the coming years with proteomics. PMID:25103677

Soybean Proteome Database 2012: Update on the comprehensive data repository for soybean proteomics

Directory of Open Access Journals (Sweden)

Hajime eOhyanagi

2012-05-01

Full Text Available The Soybean Proteome Database (SPD was created to provide a data repository for functional analyses of soybean responses to flooding stress, thought to be a major constraint for establishment and production of this plant. Since the last publication of the SPD, we thoroughly enhanced the contents of database, particularly protein samples and their annotations from several organelles. The current release contains 23 reference maps of soybean (Glycine max cv. Enrei proteins collected from several organs, tissues and organelles including the maps for plasma membrane, cell wall, chloroplast and mitochondrion, which were electrophoresed on two-dimensional polyacrylamide gels. Furthermore, the proteins analyzed with gel-free proteomics technique have been added and available online. In addition to protein fluctuations under flooding, those of salt and drought stress have been included in the current release. An omics table also has been provided to reveal relationships among mRNAs, proteins and metabolites with a unified temporal-profile tag in order to facilitate retrieval of the data based on the temporal profiles. An intuitive user interface based on dynamic HTML enables users to browse the network as well as the profiles of multiple omes in an integrated fashion. The SPD is available at: http://proteome.dc.affrc.go.jp/Soybean/.

compomics-utilities: an open-source Java library for computational proteomics.

Science.gov (United States)

Barsnes, Harald; Vaudel, Marc; Colaert, Niklaas; Helsens, Kenny; Sickmann, Albert; Berven, Frode S; Martens, Lennart

2011-03-08

The growing interest in the field of proteomics has increased the demand for software tools and applications that process and analyze the resulting data. And even though the purpose of these tools can vary significantly, they usually share a basic set of features, including the handling of protein and peptide sequences, the visualization of (and interaction with) spectra and chromatograms, and the parsing of results from various proteomics search engines. Developers typically spend considerable time and effort implementing these support structures, which detracts from working on the novel aspects of their tool. In order to simplify the development of proteomics tools, we have implemented an open-source support library for computational proteomics, called compomics-utilities. The library contains a broad set of features required for reading, parsing, and analyzing proteomics data. compomics-utilities is already used by a long list of existing software, ensuring library stability and continued support and development. As a user-friendly, well-documented and open-source library, compomics-utilities greatly simplifies the implementation of the basic features needed in most proteomics tools. Implemented in 100% Java, compomics-utilities is fully portable across platforms and architectures. Our library thus allows the developers to focus on the novel aspects of their tools, rather than on the basic functions, which can contribute substantially to faster development, and better tools for proteomics.

compomics-utilities: an open-source Java library for computational proteomics

Directory of Open Access Journals (Sweden)

Helsens Kenny

2011-03-01

Full Text Available Abstract Background The growing interest in the field of proteomics has increased the demand for software tools and applications that process and analyze the resulting data. And even though the purpose of these tools can vary significantly, they usually share a basic set of features, including the handling of protein and peptide sequences, the visualization of (and interaction with spectra and chromatograms, and the parsing of results from various proteomics search engines. Developers typically spend considerable time and effort implementing these support structures, which detracts from working on the novel aspects of their tool. Results In order to simplify the development of proteomics tools, we have implemented an open-source support library for computational proteomics, called compomics-utilities. The library contains a broad set of features required for reading, parsing, and analyzing proteomics data. compomics-utilities is already used by a long list of existing software, ensuring library stability and continued support and development. Conclusions As a user-friendly, well-documented and open-source library, compomics-utilities greatly simplifies the implementation of the basic features needed in most proteomics tools. Implemented in 100% Java, compomics-utilities is fully portable across platforms and architectures. Our library thus allows the developers to focus on the novel aspects of their tools, rather than on the basic functions, which can contribute substantially to faster development, and better tools for proteomics.

Enhancement of Environmental Hazard Degradation in the Presence of Lignin: a Proteomics Study

OpenAIRE

Sun, Su; Xie, Shangxian; Cheng, Yanbing; Yu, Hongbo; Zhao, Honglu; Li, Muzi; Li, Xiaotong; Zhang, Xiaoyu; Yuan, Joshua S.; Dai, Susie Y.

2017-01-01

Proteomics studies of fungal systems have progressed dramatically based on the availability of more fungal genome sequences in recent years. Different proteomics strategies have been applied toward characterization of fungal proteome and revealed important gene functions and proteome dynamics. Presented here is the application of shot-gun proteomic technology to study the bio-remediation of environmental hazards by white-rot fungus. Lignin, a naturally abundant component of the plant biomass,...

Advances in Proteomics of Mycobacterium leprae.

Science.gov (United States)

Parkash, O; Singh, B P

2012-04-01

Although Mycobacterium leprae was the first bacterial pathogen identified causing human disease, it remains one of the few that is non-cultivable. Understanding the biology of M. leprae is one of the primary challenges in current leprosy research. Genomics has been extremely valuable, nonetheless, functional proteins are ultimately responsible for controlling most aspects of cellular functions, which in turn could facilitate parasitizing the host. Furthermore, bacterial proteins provide targets for most of the vaccines and immunodiagnostic tools. Better understanding of the proteomics of M. leprae could also help in developing new drugs against M. leprae. During the past nearly 15 years, there have been several developments towards the identification of M. leprae proteins employing contemporary proteomics tools. In this review, we discuss the knowledge gained on the biology and pathogenesis of M. leprae from current proteomic studies. © 2012 The Authors. Scandinavian Journal of Immunology © 2012 Blackwell Publishing Ltd.

Extracellular Molecules Involved in Cancer Cell Invasion

International Nuclear Information System (INIS)

Stivarou, Theodora; Patsavoudi, Evangelia

2015-01-01

Nowadays it is perfectly clear that understanding and eradicating cancer cell invasion and metastasis represent the crucial, definitive points in cancer therapeutics. During the last two decades there has been a great interest in the understanding of the extracellular molecular mechanisms involved in cancer cell invasion. In this review, we highlight the findings concerning these processes, focusing in particular on extracellular molecules, including extracellular matrix proteins and their receptors, growth factors and their receptors, matrix metalloproteinases and extracellular chaperones. We report the molecular mechanisms underlying the important contribution of this pool of molecules to the complex, multi-step phenomenon of cancer cell invasion

Proteomic classification of breast cancer.

LENUS (Irish Health Repository)

Kamel, Dalia

2012-11-01

Being a significant health problem that affects patients in various age groups, breast cancer has been extensively studied to date. Recently, molecular breast cancer classification has advanced significantly with the availability of genomic profiling technologies. Proteomic technologies have also advanced from traditional protein assays including enzyme-linked immunosorbent assay, immunoblotting and immunohistochemistry to more comprehensive approaches including mass spectrometry and reverse phase protein lysate arrays (RPPA). The purpose of this manuscript is to review the current protein markers that influence breast cancer prediction and prognosis and to focus on novel advances in proteomic classification of breast cancer.

A model of extracellular enzymes in free-living microbes: which strategy pays off?

Science.gov (United States)

Traving, Sachia J; Thygesen, Uffe H; Riemann, Lasse; Stedmon, Colin A

2015-11-01

An initial modeling approach was applied to analyze how a single, nonmotile, free-living, heterotrophic bacterial cell may optimize the deployment of its extracellular enzymes. Free-living cells live in a dilute and complex substrate field, and to gain enough substrate, their extracellular enzymes must be utilized efficiently. The model revealed that surface-attached and free enzymes generate unique enzyme and substrate fields, and each deployment strategy has distinctive advantages. For a solitary cell, surface-attached enzymes are suggested to be the most cost-efficient strategy. This strategy entails potential substrates being reduced to very low concentrations. Free enzymes, on the other hand, generate a radically different substrate field, which suggests significant benefits for the strategy if free cells engage in social foraging or experience high substrate concentrations. Swimming has a slight positive effect for the attached-enzyme strategy, while the effect is negative for the free-enzyme strategy. The results of this study suggest that specific dissolved organic compounds in the ocean likely persist below a threshold concentration impervious to biological utilization. This could help explain the persistence and apparent refractory state of oceanic dissolved organic matter (DOM). Microbial extracellular enzyme strategies, therefore, have important implications for larger-scale processes, such as shaping the role of DOM in ocean carbon sequestration. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

MAPU: Max-Planck Unified database of organellar, cellular, tissue and body fluid proteomes.

Science.gov (United States)

Zhang, Yanling; Zhang, Yong; Adachi, Jun; Olsen, Jesper V; Shi, Rong; de Souza, Gustavo; Pasini, Erica; Foster, Leonard J; Macek, Boris; Zougman, Alexandre; Kumar, Chanchal; Wisniewski, Jacek R; Jun, Wang; Mann, Matthias

2007-01-01

Mass spectrometry (MS)-based proteomics has become a powerful technology to map the protein composition of organelles, cell types and tissues. In our department, a large-scale effort to map these proteomes is complemented by the Max-Planck Unified (MAPU) proteome database. MAPU contains several body fluid proteomes; including plasma, urine, and cerebrospinal fluid. Cell lines have been mapped to a depth of several thousand proteins and the red blood cell proteome has also been analyzed in depth. The liver proteome is represented with 3200 proteins. By employing high resolution MS and stringent validation criteria, false positive identification rates in MAPU are lower than 1:1000. Thus MAPU datasets can serve as reference proteomes in biomarker discovery. MAPU contains the peptides identifying each protein, measured masses, scores and intensities and is freely available at http://www.mapuproteome.com using a clickable interface of cell or body parts. Proteome data can be queried across proteomes by protein name, accession number, sequence similarity, peptide sequence and annotation information. More than 4500 mouse and 2500 human proteins have already been identified in at least one proteome. Basic annotation information and links to other public databases are provided in MAPU and we plan to add further analysis tools.

Integrated transcriptomic and proteomic analysis of the molecular cargo of extracellular vesicles derived from porcine adipose tissue-derived mesenchymal stem cells

OpenAIRE

Eirin, Alfonso; Zhu, Xiang-Yang; Puranik, Amrutesh S.; Woollard, John R.; Tang, Hui; Dasari, Surendra; Lerman, Amir; van Wijnen, Andre J.; Lerman, Lilach O.

2017-01-01

Background Mesenchymal stromal/stem cell (MSC) transplantation is a promising therapy for tissue regeneration. Extracellular vesicles (EVs) released by MSCs act as their paracrine effectors by delivering proteins and genetic material to recipient cells. To assess how their cargo mediates biological processes that drive their therapeutic effects, we integrated miRNA, mRNA, and protein expression data of EVs from porcine adipose tissue-derived MSCs. Methods Simultaneous expression profiles of m...

MitoMiner: a data warehouse for mitochondrial proteomics data.

Science.gov (United States)

Smith, Anthony C; Blackshaw, James A; Robinson, Alan J

2012-01-01

MitoMiner (http://mitominer.mrc-mbu.cam.ac.uk/) is a data warehouse for the storage and analysis of mitochondrial proteomics data gathered from publications of mass spectrometry and green fluorescent protein tagging studies. In MitoMiner, these data are integrated with data from UniProt, Gene Ontology, Online Mendelian Inheritance in Man, HomoloGene, Kyoto Encyclopaedia of Genes and Genomes and PubMed. The latest release of MitoMiner stores proteomics data sets from 46 studies covering 11 different species from eumetazoa, viridiplantae, fungi and protista. MitoMiner is implemented by using the open source InterMine data warehouse system, which provides a user interface allowing users to upload data for analysis, personal accounts to store queries and results and enables queries of any data in the data model. MitoMiner also provides lists of proteins for use in analyses, including the new MitoMiner mitochondrial proteome reference sets that specify proteins with substantial experimental evidence for mitochondrial localization. As further mitochondrial proteomics data sets from normal and diseased tissue are published, MitoMiner can be used to characterize the variability of the mitochondrial proteome between tissues and investigate how changes in the proteome may contribute to mitochondrial dysfunction and mitochondrial-associated diseases such as cancer, neurodegenerative diseases, obesity, diabetes, heart failure and the ageing process.

Proteomic analysis of the Arabidopsis thaliana-Botrytis cinerea ...

African Journals Online (AJOL)

A two-dimensional liquid chromatography (2D LC) system, ProteomeLab PF 2D, was employed to study the defence proteome of Arabidopsis following infection with the necrotrophic fungal pathogen, Botrytis cinerea. This system demonstrated differential protein expression in control and treated samples in some fractions.

Halobacterium salinarum NRC-1 PeptideAtlas: strategies for targeted proteomics

Science.gov (United States)

Van, Phu T.; Schmid, Amy K.; King, Nichole L.; Kaur, Amardeep; Pan, Min; Whitehead, Kenia; Koide, Tie; Facciotti, Marc T.; Goo, Young-Ah; Deutsch, Eric W.; Reiss, David J.; Mallick, Parag; Baliga, Nitin S.

2009-01-01

The relatively small numbers of proteins and fewer possible posttranslational modifications in microbes provides a unique opportunity to comprehensively characterize their dynamic proteomes. We have constructed a Peptide Atlas (PA) for 62.7% of the predicted proteome of the extremely halophilic archaeon Halobacterium salinarum NRC-1 by compiling approximately 636,000 tandem mass spectra from 497 mass spectrometry runs in 88 experiments. Analysis of the PA with respect to biophysical properties of constituent peptides, functional properties of parent proteins of detected peptides, and performance of different mass spectrometry approaches has helped highlight plausible strategies for improving proteome coverage and selecting signature peptides for targeted proteomics. Notably, discovery of a significant correlation between absolute abundances of mRNAs and proteins has helped identify low abundance of proteins as the major limitation in peptide detection. Furthermore we have discovered that iTRAQ labeling for quantitative proteomic analysis introduces a significant bias in peptide detection by mass spectrometry. Therefore, despite identifying at least one proteotypic peptide for almost all proteins in the PA, a context-dependent selection of proteotypic peptides appears to be the most effective approach for targeted proteomics. PMID:18652504

Top Down proteomics: Facts and perspectives

Energy Technology Data Exchange (ETDEWEB)

Catherman, Adam D.; Skinner, Owen S.; Kelleher, Neil L., E-mail: n-kelleher@northwestern.edu

2014-03-21

Highlights: • Top Down versus Bottom Up proteomics analysis. • Separations methods for Top Down proteomics. • Developments in mass spectrometry instrumentation and fragmentation. • Native mass spectrometry. - Abstract: The rise of the “Top Down” method in the field of mass spectrometry-based proteomics has ushered in a new age of promise and challenge for the characterization and identification of proteins. Injecting intact proteins into the mass spectrometer allows for better characterization of post-translational modifications and avoids several of the serious “inference” problems associated with peptide-based proteomics. However, successful implementation of a Top Down approach to endogenous or other biologically relevant samples often requires the use of one or more forms of separation prior to mass spectrometric analysis, which have only begun to mature for whole protein MS. Recent advances in instrumentation have been used in conjunction with new ion fragmentation using photons and electrons that allow for better (and often complete) protein characterization on cases simply not tractable even just a few years ago. Finally, the use of native electrospray mass spectrometry has shown great promise for the identification and characterization of whole protein complexes in the 100 kDa to 1 MDa regime, with prospects for complete compositional analysis for endogenous protein assemblies a viable goal over the coming few years.

Top Down proteomics: Facts and perspectives

International Nuclear Information System (INIS)

Catherman, Adam D.; Skinner, Owen S.; Kelleher, Neil L.

2014-01-01

Highlights: • Top Down versus Bottom Up proteomics analysis. • Separations methods for Top Down proteomics. • Developments in mass spectrometry instrumentation and fragmentation. • Native mass spectrometry. - Abstract: The rise of the “Top Down” method in the field of mass spectrometry-based proteomics has ushered in a new age of promise and challenge for the characterization and identification of proteins. Injecting intact proteins into the mass spectrometer allows for better characterization of post-translational modifications and avoids several of the serious “inference” problems associated with peptide-based proteomics. However, successful implementation of a Top Down approach to endogenous or other biologically relevant samples often requires the use of one or more forms of separation prior to mass spectrometric analysis, which have only begun to mature for whole protein MS. Recent advances in instrumentation have been used in conjunction with new ion fragmentation using photons and electrons that allow for better (and often complete) protein characterization on cases simply not tractable even just a few years ago. Finally, the use of native electrospray mass spectrometry has shown great promise for the identification and characterization of whole protein complexes in the 100 kDa to 1 MDa regime, with prospects for complete compositional analysis for endogenous protein assemblies a viable goal over the coming few years

Analytical performance of reciprocal isotope labeling of proteome digests for quantitative proteomics and its application for comparative studies of aerobic and anaerobic Escherichia coli proteomes

International Nuclear Information System (INIS)

Lo, Andy; Weiner, Joel H.; Li, Liang

2013-01-01

Graphical abstract: -- Highlights: •Investigating a strategy of reciprocal isotope labeling of comparative samples. •Filtering out incorrect peptide identification or quantification values. •Analyzing the proteome changes of E. coli cells grown aerobically or anaerobically. •Presenting guidelines for reciprocal labeling experimental design. -- Abstract: Due to limited sample amounts, instrument time considerations, and reagent costs, only a small number of replicate experiments are typically performed for quantitative proteome analyses. Generation of reproducible data that can be readily assessed for consistency within a small number of datasets is critical for accurate quantification. We report our investigation of a strategy using reciprocal isotope labeling of two comparative samples as a tool for determining proteome changes. Reciprocal labeling was evaluated to determine the internal consistency of quantified proteome changes from Escherichia coli grown under aerobic and anaerobic conditions. Qualitatively, the peptide overlap between replicate analyses of the same sample and reverse labeled samples were found to be within 8%. Quantitatively, reciprocal analyses showed only a slight increase in average overall inconsistency when compared with replicate analyses (1.29 vs. 1.24-fold difference). Most importantly, reverse labeling was successfully used to identify spurious values resulting from incorrect peptide identifications and poor peak fitting. After removal of 5% of the peptide data with low reproducibility, a total of 275 differentially expressed proteins (>1.50-fold difference) were consistently identified and were then subjected to bioinformatics analysis. General considerations and guidelines for reciprocal labeling experimental design and biological significance of obtained results are discussed

Extracellular vesicles: Exosomes, microvesicles, and friends

NARCIS (Netherlands)

Raposo, G.; Stoorvogel, W.|info:eu-repo/dai/nl/074352385

2013-01-01

Cells release into the extracellular environment diverse types of membrane vesicles of endosomal and plasma membrane origin called exosomes and microvesicles, respectively. These extracellular vesicles (EVs) represent an important mode of intercellular communication by serving as vehicles for

The planar cell polarity protein VANGL2 coordinates remodeling of the extracellular matrix.

Science.gov (United States)

Williams, B Blairanne; Mundell, Nathan; Dunlap, Julie; Jessen, Jason

2012-07-01

Understanding how planar cell polarity (PCP) is established, maintained, and coordinated in migrating cell populations is an important area of research with implications for both embryonic morphogenesis and tumor cell invasion. We recently reported that the PCP protein Vang-like 2 (VANGL2) regulates the endocytosis and cell surface level of membrane type-1 matrix metalloproteinase (MMP14 or MT1-MMP). Here, we further discuss these findings in terms of extracellular matrix (ECM) remodeling, cell migration, and zebrafish gastrulation. We also demonstrate that VANGL2 function impacts the focal degradation of ECM by human cancer cells including the formation or stability of invadopodia. Together, our findings implicate MMP14 as a downstream effector of VANGL2 signaling and suggest a model whereby the regulation of pericellular proteolysis is a fundamental aspect of PCP in migrating cells.

C4 photosynthetic machinery: insights from maize chloroplast proteomics

Directory of Open Access Journals (Sweden)

Qi eZhao

2013-04-01

Full Text Available C4 plants exhibit much higher CO2 assimilation rates than C3 plants. The specialized differentiation of mesophyll cell (M and bundle sheath cell (BS type chloroplasts is unique to C4 plants and improves photosynthesis efficiency. Maize (Zea mays is an important crop and model with C4 photosynthetic machinery. Current high-throughput quantitative proteomics approaches (e.g., 2DE, iTRAQ, and shotgun proteomics have been employed to investigate maize chloroplast structure and function. These proteomic studies have provided valuable information on C4 chloroplast protein components, photosynthesis, and other metabolic mechanisms underlying chloroplast biogenesis, stromal and membrane differentiation, as well as response to salinity, high/low temperature, and light stress. This review presents an overview of proteomics advances in maize chloroplast biology.

Extracellular miRNA-21 as a novel biomarker in glioma: evidence from meta-analysis, clinical validation and experimental investigations

Science.gov (United States)

Liu, Tian; Wang, Zhixin; Tai, Minghui; Meng, Fandi; Zhang, Jingyao; Wan, Yong; Mao, Ping; Dong, Xiaoqun; Liu, Chang; Niu, Wenquan; Dong, Shunbin

2016-01-01

Evidence is accumulating highlighting the importance of extracellular miRNA as a novel biomarker for diagnosing various kinds of malignancies. MiR-21 is one of the most studied miRNAs and is over-expressed in cancer tissues. To explore the clinical implications and secretory mechanisms of extracellular miR-21, we firstly meta-analyzed the diagnostic efficiency of extracellular miR-21 in different cancer types. Eighty-one studies based on 59 articles were finally included. In our study, extracellular miR-21 was observed to exhibit an outstanding diagnostic accuracy in detecting brain cancer (area under the summary receiver operating characteristic curve or AUC = 0.94), and this accuracy was more obvious in glioma diagnosis (AUC = 0.95). Our validation study (n = 45) further confirmed the diagnostic and prognostic role of miR-21 in cerebrospinal fluid (CSF) for glioma. These findings inspired us to explore the biological function of miR-21. We next conducted mechanistic investigations to explain the secretory mechanisms of extracellular miR-21 in glioma. TGF-β/Smad3 signaling was identified to participate in mediating the release of miR-21 from glioma cells. Further targeting TGF-β/Smad3 signaling using galunisertib, an inhibitor of the TGF-β type I receptor kinase, can attenuate the secretion of miR-21 from glioma cells. Taken together, CSF-based miR-21 might serve as a potential biomarker for diagnosing brain cancer, especially for patients with glioma. Moreover, extracellular levels of miR-21 were affected by exogenous TGF-β activity and galunisertib treatment. PMID:27166186

New insights into the mechanisms of acetic acid resistance in Acetobacter pasteurianus using iTRAQ-dependent quantitative proteomic analysis.

Science.gov (United States)

Xia, Kai; Zang, Ning; Zhang, Junmei; Zhang, Hong; Li, Yudong; Liu, Ye; Feng, Wei; Liang, Xinle

2016-12-05

Acetobacter pasteurianus is the main starter in rice vinegar manufacturing due to its remarkable abilities to resist and produce acetic acid. Although several mechanisms of acetic acid resistance have been proposed and only a few effector proteins have been identified, a comprehensive depiction of the biological processes involved in acetic acid resistance is needed. In this study, iTRAQ-based quantitative proteomic analysis was adopted to investigate the whole proteome of different acidic titers (3.6, 7.1 and 9.3%, w/v) of Acetobacter pasteurianus Ab3 during the vinegar fermentation process. Consequently, 1386 proteins, including 318 differentially expressed proteins (p150 proteins were differentially expressed. Specifically, proteins involved in amino acid metabolic processes and fatty acid biosynthesis were differentially expressed, which may contribute to the acetic acid resistance of Acetobacter. Transcription factors, two component systems and toxin-antitoxin systems were implicated in the modulatory network at multiple levels. In addition, the identification of proteins involved in redox homeostasis, protein metabolism, and the cell envelope suggested that the whole cellular system is mobilized in response to acid stress. These findings provide a differential proteomic profile of acetic acid resistance in Acetobacter pasteurianus and have potential application to highly acidic rice vinegar manufacturing. Copyright © 2016 Elsevier B.V. All rights reserved.

Dark production of extracellular superoxide by the coral Porites astreoides and representative symbionts

Directory of Open Access Journals (Sweden)

Tong Zhang

2016-11-01

Full Text Available The reactive oxygen species (ROS superoxide has been implicated in both beneficial and detrimental processes in coral biology, ranging from pathogenic disease resistance to coral bleaching. Despite the critical role of ROS in coral health, there is a distinct lack of ROS measurements and thus an incomplete understanding of underpinning ROS sources and production mechanisms within coral systems. Here, we quantified in situ extracellular superoxide concentrations at the surfaces of aquaria-hosted Porites astreoides during a diel cycle. High concentrations of superoxide (~10’s of nM were present at coral surfaces, and these levels did not change significantly as a function of time of day. These results indicate that the coral holobiont produces extracellular superoxide in the dark, independent of photosynthesis. As a short-lived anion at physiological pH, superoxide has a limited ability to cross intact biological membranes. Further, removing surface mucus layers from the P. astreoides colonies did not impact external superoxide concentrations. We therefore attribute external superoxide derived from the coral holobiont under these conditions to the activity of the coral host epithelium, rather than mucus-derived epibionts or internal sources such as endosymbionts (e.g., Symbiodinium. However, endosymbionts likely contribute to internal ROS levels via extracellular superoxide production. Indeed, common coral symbionts, including multiple strains of Symbiodinium (clades A to D and the bacterium Endozoicomonas montiporae LMG 24815, produced extracellular superoxide in the dark and at low light levels. Further, representative P. astreoides symbionts, Symbiodinium CCMP2456 (clade A and E. montiporae, produced similar concentrations of superoxide alone and in combination with each other, in the dark and low light, and regardless of time of day. Overall, these results indicate that healthy, non-stressed P. astreoides and representative symbionts produce

The 3rd Central and Eastern European Proteomic Conference

Czech Academy of Sciences Publication Activity Database

Gadher, S. J.; Martinková, Jiřina; Drahoš, L.; Vékey, K.; Allmaier, G.; Kovářová, Hana

2010-01-01

Roč. 7, č. 1 (2010), s. 15-17 ISSN 1478-9450 Institutional research plan: CEZ:AV0Z50450515 Keywords : proteomics * proteome research * biomarkers Subject RIV: CE - Biochemistry Impact factor: 4.406, year: 2010

Sources of extracellular tau and its signaling.

Science.gov (United States)

Avila, Jesús; Simón, Diana; Díaz-Hernández, Miguel; Pintor, Jesús; Hernández, Félix

2014-01-01

The pathology associated with tau protein, tauopathy, has been recently analyzed in different disorders, leading to the suggestion that intracellular and extracellular tau may itself be the principal agent in the transmission and spreading of tauopathies. Tau pathology is based on an increase in the amount of tau, an increase in phosphorylated tau, and/or an increase in aggregated tau. Indeed, phosphorylated tau protein is the main component of tau aggregates, such as the neurofibrillary tangles present in the brain of Alzheimer's disease patients. It has been suggested that intracellular tau could be toxic to neurons in its phosphorylated and/or aggregated form. However, extracellular tau could also damage neurons and since neuronal death is widespread in Alzheimer's disease, mainly among cholinergic neurons, these cells may represent a possible source of extracellular tau. However, other sources of extracellular tau have been proposed that are independent of cell death. In addition, several ways have been proposed for cells to interact with, transmit, and spread extracellular tau, and to transduce signals mediated by this tau. In this work, we will discuss the role of extracellular tau in the spreading of the tau pathology.

Extracellular matrix assembly in extreme acidic eukaryotic biofilms and their possible implications in heavy metal adsorption

Energy Technology Data Exchange (ETDEWEB)

Aguilera, Angeles [Centro de Astrobiologia (INTA-CSIC), Carretera de Ajalvir Km 4, Torrejon de Ardoz, 28850 Madrid (Spain)], E-mail: aguileraba@inta.es; Souza-Egipsy, Virginia [Centro de Astrobiologia (INTA-CSIC), Carretera de Ajalvir Km 4, Torrejon de Ardoz, 28850 Madrid (Spain); San Martin-Uriz, Patxi [Centro de Biologia Molecular (UAM-CSIC), Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain); Amils, Ricardo [Centro de Astrobiologia (INTA-CSIC), Carretera de Ajalvir Km 4, Torrejon de Ardoz, 28850 Madrid (Spain); Centro de Biologia Molecular (UAM-CSIC), Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain)

2008-07-30

To evaluate the importance of the extracellular matrix in relation to heavy metal binding capacity in extreme acidic environments, the extracellular polymeric substances (EPS) composition of 12 biofilms isolated from Rio Tinto (SW, Spain) was analyzed. Each biofilm was composed mainly by one or two species of eukaryotes, although other microorganisms were present. EPS ranged from 130 to 439 mg g{sup -1} biofilm dry weight, representing between 15% and the 40% of the total biofilm dry weight (DW). Statistically significant differences (p < 0.05) were found in the amount of total EPS extracted from biofilms dominated by the same organism at different sampling points. The amount of EPS varied among different biofilms collected from the same sampling location. Colloidal EPS ranged from 42 to 313 mg g{sup -1} dry weight; 10% to 30% of the total biofilm dry weight. Capsular EPS ranged from 50 to 318 mg g{sup -1} dry weight; 5% to 30% of the total biofilm dry weight. Seven of the 12 biofilms showed higher amounts of capsular than colloidal EPS (p < 0.05). Total amount of EPS decreased when total cell numbers and pH increased. There was a positive correlation between EPS concentration and heavy metal concentration in the water. Observations by low temperature scanning electron microscopy (LTSEM) revealed the mineral adsorption in the matrix of EPS and onto the cell walls. EPS in all biofilms were primarily composed of carbohydrates, heavy metals and humic acid, plus small quantities of proteins and DNA. After carbohydrates, heavy metals were the second main constituents of the extracellular matrix. Their total concentrations ranged from 3 to 32 mg g{sup -1} biofilm dry weight, reaching up to 16% of the total composition. In general, the heavy metal composition of the EPS extracted from the biofilms closely resembled the metal composition of the water from which the biofilms were collected.

Analysis of extracellular RNA by digital PCR

Directory of Open Access Journals (Sweden)

Kenji eTakahashi

2014-06-01

Full Text Available The transfer of extracellular RNA is emerging as an important mechanism for intracellular communication. The ability for the transfer of functionally active RNA molecules from one cell to another within vesicles such as exosomes enables a cell to modulate cellular signaling and biological processes within recipient cells. The study of extracellular RNA requires sensitive methods for the detection of these molecules. In this methods article, we will describe protocols for the detection of such extracellular RNA using sensitive detection technologies such as digital PCR. These protocols should be valuable to researchers interested in the role and contribution of extracellular RNA to tumor cell biology.

Extracellular Molecules Involved in Cancer Cell Invasion

Directory of Open Access Journals (Sweden)

Theodora Stivarou

2015-01-01

Full Text Available Nowadays it is perfectly clear that understanding and eradicating cancer cell invasion and metastasis represent the crucial, definitive points in cancer therapeutics. During the last two decades there has been a great interest in the understanding of the extracellular molecular mechanisms involved in cancer cell invasion. In this review, we highlight the findings concerning these processes, focusing in particular on extracellular molecules, including extracellular matrix proteins and their receptors, growth factors and their receptors, matrix metalloproteinases and extracellular chaperones. We report the molecular mechanisms underlying the important contribution of this pool of molecules to the complex, multi-step phenomenon of cancer cell invasion.

Biophysical characterization of the olfactomedin domain of myocilin, an extracellular matrix protein implicated in inherited forms of glaucoma.

Directory of Open Access Journals (Sweden)

Susan D Orwig

Full Text Available Myocilin is an eye protein found in the trabecular extracellular matrix (TEM, within the anatomic region that controls fluid flow. Variants of myocilin, localized to its olfactomedin (OLF domain, have been linked to inherited forms of glaucoma, a disease associated with elevated intraocular pressure. OLF domains have also been implicated in psychiatric diseases and cancers by their involvement in signaling, neuronal growth, and development. However, molecular characterization of OLFs has been hampered by challenges in recombinant expression, a hurdle we have recently overcome for the myocilin OLF domain (myoc-OLF. Here, we report the first detailed solution biophysical characterization of myoc-OLF to gain insight into its structure and function. Myoc-OLF is stable in the presence of glycosaminoglycans, as well as in a wide pH range in buffers with functional groups reminiscent of such glycosaminoglycans. Circular dichroism (CD reveals significant β-sheet and β-turn secondary structure. Unexpectedly, the CD signature is reminiscent of α-chymotrypsin as well as another ocular protein family, the βγ-crystallins. At neutral pH, intrinsic tryptophan fluorescence and CD melts indicate a highly cooperative transition with a melting temperature of ∼55 °C. Limited proteolysis combined with mass spectrometry reveals that the compact core structural domain of OLF consists of approximately residues 238-461, which retains the single disulfide bond and is as stable as the full myoc-OLF construct. The data presented here inform new testable hypotheses for interactions with specific TEM components, and will assist in design of therapeutic agents for myocilin glaucoma.

Cutting edge proteomics

DEFF Research Database (Denmark)

Bunkenborg, Jakob; Espadas, Guadalupe; Molina, Henrik

2013-01-01

Tryptic digestion is an important component of most proteomics experiments, and trypsin is available from many sources with a cost that varies by more than 1000-fold. This high-mass-accuracy LC-MS study benchmarks six commercially available trypsins with respect to autolytic species and sequence ...

A Proposal of the Ur-proteome

Science.gov (United States)

Palacios-Pérez, Miryam; Andrade-Díaz, Fernando; José, Marco V.

2017-11-01

Herein we outline a plausible proteome, encoded by assuming a primeval RNY genetic code. We unveil the primeval phenotype by using only the RNA genotype; it means that we recovered the most ancestral proteome, mostly made of the 8 amino acids encoded by RNY triplets. By looking at those fragments, it is noticeable that they are positioned, not at catalytic sites, but in the cofactor binding sites. It implies that the stabilization of a molecule appeared long before its catalytic activity, and therefore the Ur-proteome comprised a set of proteins modules that corresponded to Cofactor Stabilizing Binding Sites (CSBSs), which we call the primitive bindome. With our method, we reconstructed the structures of the "first protein modules" that Sobolevsky and Trifonov (2006) found by using only RMSD. We also examine the probable cofactors that bound to them. We discuss the notion of CSBSs as the first proteins modules in progenotes in the context of several proposals about the primitive forms of life.

Phenotypic and Proteomic Analysis of the Aspergillus fumigatus ΔPrtT, ΔXprG and ΔXprG/ΔPrtT Protease-Deficient Mutants

Directory of Open Access Journals (Sweden)

Einav Shemesh

2017-12-01

Full Text Available Aspergillus fumigatus is the most common mold species to cause disease in immunocompromised patients. Infection usually begins when its spores (conidia are inhaled into the airways, where they germinate, forming hyphae that penetrate and destroy the lungs and disseminate to other organs, leading to high mortality. The ability of hyphae to penetrate the pulmonary epithelium is a key step in the infectious process. A. fumigatus produces extracellular proteases that are thought to enhance penetration by degrading host structural barriers. This study explores the role of the A. fumigatus transcription factor XprG in controlling secreted proteolytic activity and fungal virulence. We deleted xprG, alone and in combination with prtT, a transcription factor previously shown to regulate extracellular proteolysis. xprG deletion resulted in abnormal conidiogenesis and formation of lighter colored, more fragile conidia and a moderate reduction in the ability of culture filtrates (CFs to degrade substrate proteins. Deletion of both xprG and prtT resulted in an additive reduction, generating a mutant strain producing CF with almost no ability to degrade substrate proteins. Detailed proteomic analysis identified numerous secreted proteases regulated by XprG and PrtT, alone and in combination. Interestingly, proteomics also identified reduced levels of secreted cell wall modifying enzymes (glucanases, chitinases and allergens following deletion of these genes, suggesting they target additional cellular processes. Surprisingly, despite the major alteration in the secretome of the xprG/prtT null mutant, including two to fivefold reductions in the level of 24 proteases, 18 glucanases, 6 chitinases, and 19 allergens, it retained wild-type virulence in murine systemic and pulmonary models of infection. This study highlights the extreme adaptability of A. fumigatus during infection based on extensive gene redundancy.

[Inhibitory proteins of neuritic regeneration in the extracellular matrix: structure, molecular interactions and their functions. Mechanisms of extracellular balance].

Science.gov (United States)

Vargas, Javier; Uribe-Escamilla, Rebeca; Alfaro-Rodríguez, Alfonso

2013-01-01

After injury of the central nervous system (CNS) in higher vertebrates, neurons neither grow nor reconnect with their targets because their axons or dendrites cannot regenerate within the injured site. In the CNS, the signal from the environment regulating neurite regeneration is not exclusively generated by one molecular group. This signal is generated by the interaction of various types of molecules such as extracellular matrix proteins, soluble factors and surface membrane molecules; all these elements interact with one another generating the matrix's biological state: the extracellular balance. Proteins in the balanced extracellular matrix, support and promote cellular physiological states, including neuritic regeneration. We have reviewed three types of proteins of the extracellular matrix possessing an inhibitory effect and that are determinant of neuritic regeneration failure in the CNS: chondroitin sulfate proteoglycans, keratan sulfate proteoglycans and tenascin. We also review some of the mechanisms involved in the balance of extracellular proteins such as isomerization, epimerization, sulfation and glycosylation as well as the assemblage of the extracellular matrix, the interaction between the matrix and soluble factors and its proteolytic degradation. In the final section, we have presented some examples of the matrix's role in development and in tumor propagation.

Variation in bacterial ATP concentration during rapid changes in extracellular pH and implications for the activity of attached bacteria.

Science.gov (United States)

Albert, Lynal S; Brown, Derick G

2015-08-01

In this study we investigated the relationship between a rapid change in extracellular pH and the alteration of bacterial ATP concentration. This relationship is a key component of a hypothesis indicating that bacterial bioenergetics - the creation of ATP from ADP via a proton gradient across the cytoplasmic membrane - can be altered by the physiochemical charge-regulation effect, which results in a pH shift at the bacteria's surface upon adhesion to another surface. The bacterial ATP concentration was measured during a rapid change in extracellular pH from a baseline pH of 7.2 to pH values between 3.5 and 10.5. Experiments were conducted with four neutrophilic bacterial strains, including the Gram-negative Escherichia coli and Pseudomonas putida and the Gram-positive Bacillus subtilis and Staphylococcus epidermidis. A change in bulk pH produced an immediate response in bacterial ATP, demonstrating a direct link between changes in extracellular pH and cellular bioenergetics. In general, the shifts in ATP were similar across the four bacterial strains, with results following an exponential relationship between the extracellular pH and cellular ATP concentration. One exception occurred with S. epidermidis, where there was no variation in cellular ATP at acidic pH values, and this finding is consistent with this species' ability to thrive under acidic conditions. These results provide insight into obtaining a desired bioenergetic response in bacteria through (i) the application of chemical treatments to vary the local pH and (ii) the selection and design of surfaces resulting in local pH modification of attached bacteria via the charge-regulation effect. Copyright © 2015 Elsevier B.V. All rights reserved.

Proteomics reveals multiple routes to the osteogenic phenotype in mesenchymal stem cells

Directory of Open Access Journals (Sweden)

Yener Bülent

2007-10-01

Full Text Available Abstract Background Recently, we demonstrated that human mesenchymal stem cells (hMSC stimulated with dexamethazone undergo gene focusing during osteogenic differentiation (Stem Cells Dev 14(6: 1608–20, 2005. Here, we examine the protein expression profiles of three additional populations of hMSC stimulated to undergo osteogenic differentiation via either contact with pro-osteogenic extracellular matrix (ECM proteins (collagen I, vitronectin, or laminin-5 or osteogenic media supplements (OS media. Specifically, we annotate these four protein expression profiles, as well as profiles from naïve hMSC and differentiated human osteoblasts (hOST, with known gene ontologies and analyze them as a tensor with modes for the expressed proteins, gene ontologies, and stimulants. Results Direct component analysis in the gene ontology space identifies three components that account for 90% of the variance between hMSC, osteoblasts, and the four stimulated hMSC populations. The directed component maps the differentiation stages of the stimulated stem cell populations along the differentiation axis created by the difference in the expression profiles of hMSC and hOST. Surprisingly, hMSC treated with ECM proteins lie closer to osteoblasts than do hMSC treated with OS media. Additionally, the second component demonstrates that proteomic profiles of collagen I- and vitronectin-stimulated hMSC are distinct from those of OS-stimulated cells. A three-mode tensor analysis reveals additional focus proteins critical for characterizing the phenotypic variations between naïve hMSC, partially differentiated hMSC, and hOST. Conclusion The differences between the proteomic profiles of OS-stimulated hMSC and ECM-hMSC characterize different transitional phenotypes en route to becoming osteoblasts. This conclusion is arrived at via a three-mode tensor analysis validated using hMSC plated on laminin-5.

Rabbit seminal plasma proteome: The importance of the genetic origin.

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Casares-Crespo, Lucía; Fernández-Serrano, Paula; Vicente, José S; Marco-Jiménez, Francisco; Viudes-de-Castro, María Pilar

2018-02-01

The present study was conducted to characterise rabbit seminal plasma proteins (SP proteins) focusing on the influence of the genetic origin and seasonality. In addition, β-NGF protein quantity in SP was determined. Semen samples were recovered from January to December 2014 using 6 males belonging to genotype A and six from genotype R. For each genotype, one pooled sample at the beginning, middle and end of each season was selected to develop the experiment. A total of 24 pools (3 for each season and genetic line) were analysed. SP proteins of the two experimental groups were recovered and subjected to in-solution digestion nano LC-MS/MS and bioinformatics analysis. The resulting library included 402 identified proteins validated with ≥95% Confidence (unused Score ≥ 1.3). These data are available via ProteomeXchange with identifier PXD006308. Only 6 proteins were specifically implicated in reproductive processes according to Gene Ontology annotation. Twenty-three proteins were differentially expressed between genotypes, 11 over-expressed in genotype A and 12 in genotype R. Regarding the effect of season on rabbit SP proteome, results showed that there is no clear pattern of protein variation throughout the year. Similar β-NGF relative quantity was observed between seasons and genotypes. In conclusion, this study generates the largest library of SP proteins reported to date in rabbits and provides evidence that genotype is related to a specific abundance of SP proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

Proteomics of Rice Seed Germination

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Dongli eHe

2013-07-01

Full Text Available Seed is a condensed form of plant. Under suitable environmental conditions, it can resume the metabolic activity from physiological quiescent status, and mobilize the reserves, biosynthesize new proteins, regenerate organelles and cell membrane, eventually protrude the radicle and enter into seedling establishment. So far, how these activities are regulated in a coordinated and sequential manner is largely unknown. With the availability of more and more genome sequence information and the development of mass spectrometry (MS technology, proteomics has been widely applied in analyzing the mechanisms of different biological processes, and proved to be very powerful. Regulation of rice seed germination is critical for rice cultivation. In recent years, a lot of proteomic studies have been conducted in exploring the gene expression regulation, reserves mobilization and metabolisms reactivation, which brings us new insights on the mechanisms of metabolism regulation during this process. Nevertheless, it also invokes a lot of questions. In this mini-review, we summarized the progress in the proteomic studies of rice seed germination. The current challenges and future perspectives were also discussed, which might be helpful for the following studies.

Proteomic identification of gender molecular markers in Bothrops jararaca venom.

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Zelanis, André; Menezes, Milene C; Kitano, Eduardo S; Liberato, Tarcísio; Tashima, Alexandre K; Pinto, Antonio F M; Sherman, Nicholas E; Ho, Paulo L; Fox, Jay W; Serrano, Solange M T

2016-04-29

Variation in the snake venom proteome is a well-documented phenomenon; however, sex-based variation in the venom proteome/peptidome is poorly understood. Bothrops jararaca shows significant sexual size dimorphism and here we report a comparative proteomic/peptidomic analysis of venoms from male and female specimens and correlate it with the evaluation of important venom features. We demonstrate that adult male and female venoms have distinct profiles of proteolytic activity upon fibrinogen and gelatin. These differences were clearly reflected in their different profiles of SDS-PAGE, two-dimensional electrophoresis and glycosylated proteins. Identification of differential protein bands and spots between male or female venoms revealed gender-specific molecular markers. However, the proteome comparison by in-solution trypsin digestion and label-free quantification analysis showed that the overall profiles of male and female venoms are similar at the polypeptide chain level but show striking variation regarding their attached carbohydrate moieties. The analysis of the peptidomes of male and female venoms revealed different contents of peptides, while the bradykinin potentiating peptides (BPPs) showed rather similar profiles. Furthermore we confirmed the ubiquitous presence of four BPPs that lack the C-terminal Q-I-P-P sequence only in the female venom as gender molecular markers. As a result of these studies we demonstrate that the sexual size dimorphism is associated with differences in the venom proteome/peptidome in B. jararaca species. Moreover, gender-based variations contributed by different glycosylation levels in toxins impact venom complexity. Bothrops jararaca is primarily a nocturnal and generalist snake species, however, it exhibits a notable ontogenetic shift in diet and in venom proteome upon neonate to adult transition. As is common in the Bothrops genus, B. jararaca shows significant sexual dimorphism in snout-vent length and weight, with females being

Proteomics Analysis of Cancer Exosomes Using a Novel Modified Aptamer-based Array (SOMAscanTM) Platform*

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Webber, Jason; Stone, Timothy C.; Katilius, Evaldas; Smith, Breanna C.; Gordon, Bridget; Mason, Malcolm D.; Tabi, Zsuzsanna; Brewis, Ian A.; Clayton, Aled

2014-01-01

We have used a novel affinity-based proteomics technology to examine the protein signature of small secreted extracellular vesicles called exosomes. The technology uses a new class of protein binding reagents called SOMAmers® (slow off-rate modified aptamers) and allows the simultaneous precise measurement of over 1000 proteins. Exosomes were highly purified from the Du145 prostate cancer cell line, by pooling selected fractions from a continuous sucrose gradient (within the density range of 1.1 to 1.2 g/ml), and examined under standard conditions or with additional detergent treatment by the SOMAscanTM array (version 3.0). Lysates of Du145 cells were also prepared, and the profiles were compared. Housekeeping proteins such as cyclophilin-A, LDH, and Hsp70 were present in exosomes, and we identified almost 100 proteins that were enriched in exosomes relative to cells. These included proteins of known association with cancer exosomes such as MFG-E8, integrins, and MET, and also those less widely reported as exosomally associated, such as ROR1 and ITIH4. Several proteins with no previously known exosomal association were confirmed as exosomally expressed in experiments using individual SOMAmer® reagents or antibodies in micro-plate assays. Western blotting confirmed the SOMAscanTM-identified enrichment of exosomal NOTCH-3, L1CAM, RAC1, and ADAM9. In conclusion, we describe here over 300 proteins of hitherto unknown association with prostate cancer exosomes and suggest that the SOMAmer®-based assay technology is an effective proteomics platform for exosome-associated biomarker discovery in diverse clinical settings. PMID:24505114

TAILS N-terminomic and proteomic datasets of healthy human dental pulp

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Ulrich Eckhard

2015-12-01

Full Text Available The Data described here provide the in depth proteomic assessment of the human dental pulp proteome and N-terminome (Eckhard et al., 2015 [1]. A total of 9 human dental pulps were processed and analyzed by the positional proteomics technique TAILS (Terminal Amine Isotopic Labeling of Substrates N-terminomics. 38 liquid chromatography tandem mass spectrometry (LC-MS/MS datasets were collected and analyzed using four database search engines in combination with statistical downstream evaluation, to yield the by far largest proteomic and N-terminomic dataset of any dental tissue to date. The raw mass spectrometry data and the corresponding metadata have been deposited in ProteomeXchange with the PXD identifier ; Supplementary Tables described in this article are available via Mendeley Data (10.17632/555j3kk4sw.1.

Extracellular histones in tissue injury and inflammation.

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Allam, Ramanjaneyulu; Kumar, Santhosh V R; Darisipudi, Murthy N; Anders, Hans-Joachim

2014-05-01

Neutrophil NETosis is an important element of host defense as it catapults chromatin out of the cell to trap bacteria, which then are killed, e.g., by the chromatin's histone component. Also, during sterile inflammation TNF-alpha and other mediators trigger NETosis, which elicits cytotoxic effects on host cells. The same mechanism should apply to other forms of regulated necrosis including pyroptosis, necroptosis, ferroptosis, and cyclophilin D-mediated regulated necrosis. Beyond these toxic effects, extracellular histones also trigger thrombus formation and innate immunity by activating Toll-like receptors and the NLRP3 inflammasome. Thereby, extracellular histones contribute to the microvascular complications of sepsis, major trauma, small vessel vasculitis as well as acute liver, kidney, brain, and lung injury. Finally, histones prevent the degradation of extracellular DNA, which promotes autoimmunization, anti-nuclear antibody formation, and autoimmunity in susceptible individuals. Here, we review the current evidence on the pathogenic role of extracellular histones in disease and discuss how to target extracellular histones to improve disease outcomes.

Proteomic analysis of proteins expressing in regions of rat brain by a combination of SDS-PAGE with nano-liquid chromatography-quadrupole-time of flight tandem mass spectrometry

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Maekawa Tsuyoshi

2010-07-01

Full Text Available Abstract Background Most biological functions controlled by the brain and their related disorders are closely associated with activation in specific regions of the brain. Neuroproteomics has been applied to the analysis of whole brain, and the general pattern of protein expression in all regions has been elucidated. However, the comprehensive proteome of each brain region remains unclear. Results In this study, we carried out comparative proteomics of six regions of the adult rat brain: thalamus, hippocampus, frontal cortex, parietal cortex, occipital cortex, and amygdala using semi-quantitative analysis by Mascot Score of the identified proteins. In order to identify efficiently the proteins that are present in the brain, the proteins were separated by a combination of SDS-PAGE on a C18 column-equipped nano-liquid chromatograph, and analyzed by quadrupole-time of flight-tandem-mass spectrometry. The proteomic data show 2,909 peptides in the rat brain, with more than 200 identified as region-abundant proteins by semi-quantitative analysis. The regions containing the identified proteins are membrane (20.0%, cytoplasm (19.5%, mitochondrion (17.1%, cytoskeleton (8.2%, nucleus (4.7%, extracellular region (3.3%, and other (18.0%. Of the identified proteins, the expressions of glial fibrillary acidic protein, GABA transporter 3, Septin 5, heat shock protein 90, synaptotagmin, heat shock protein 70, and pyruvate kinase were confirmed by immunoblotting. We examined the distributions in rat brain of GABA transporter 3, glial fibrillary acidic protein, and heat shock protein 70 by immunohistochemistry, and found that the proteins are localized around the regions observed by proteomic analysis and immunoblotting. IPA analysis indicates that pathways closely related to the biological functions of each region may be activated in rat brain. Conclusions These observations indicate that proteomics in each region of adult rat brain may provide a novel way to

PROTEINCHALLENGE: Crowd sourcing in proteomics analysis and software development

DEFF Research Database (Denmark)

Martin, Sarah F.; Falkenberg, Heiner; Dyrlund, Thomas Franck

2013-01-01

, including arguments for community-wide open source software development and “big data” compatible solutions for the future. For the meantime, we have laid out ten top tips for data processing. With these at hand, a first large-scale proteomics analysis hopefully becomes less daunting to navigate.......However there is clearly a real need for robust tools, standard operating procedures and general acceptance of best practises. Thus we submit to the proteomics community a call for a community-wide open set of proteomics analysis challenges—PROTEINCHALLENGE—that directly target and compare data analysis workflows......In large-scale proteomics studies there is a temptation, after months of experimental work, to plug resulting data into a convenient—if poorly implemented—set of tools, which may neither do the data justice nor help answer the scientific question. In this paper we have captured key concerns...

Parasites, proteomes and systems: has Descartes' clock run out of time?

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Wastling, J M; Armstrong, S D; Krishna, R; Xia, D

2012-08-01

Systems biology aims to integrate multiple biological data types such as genomics, transcriptomics and proteomics across different levels of structure and scale; it represents an emerging paradigm in the scientific process which challenges the reductionism that has dominated biomedical research for hundreds of years. Systems biology will nevertheless only be successful if the technologies on which it is based are able to deliver the required type and quality of data. In this review we discuss how well positioned is proteomics to deliver the data necessary to support meaningful systems modelling in parasite biology. We summarise the current state of identification proteomics in parasites, but argue that a new generation of quantitative proteomics data is now needed to underpin effective systems modelling. We discuss the challenges faced to acquire more complete knowledge of protein post-translational modifications, protein turnover and protein-protein interactions in parasites. Finally we highlight the central role of proteome-informatics in ensuring that proteomics data is readily accessible to the user-community and can be translated and integrated with other relevant data types.

Proteomics Standards Initiative: Fifteen Years of Progress and Future Work.

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Deutsch, Eric W; Orchard, Sandra; Binz, Pierre-Alain; Bittremieux, Wout; Eisenacher, Martin; Hermjakob, Henning; Kawano, Shin; Lam, Henry; Mayer, Gerhard; Menschaert, Gerben; Perez-Riverol, Yasset; Salek, Reza M; Tabb, David L; Tenzer, Stefan; Vizcaíno, Juan Antonio; Walzer, Mathias; Jones, Andrew R

2017-12-01

The Proteomics Standards Initiative (PSI) of the Human Proteome Organization (HUPO) has now been developing and promoting open community standards and software tools in the field of proteomics for 15 years. Under the guidance of the chair, cochairs, and other leadership positions, the PSI working groups are tasked with the development and maintenance of community standards via special workshops and ongoing work. Among the existing ratified standards, the PSI working groups continue to update PSI-MI XML, MITAB, mzML, mzIdentML, mzQuantML, mzTab, and the MIAPE (Minimum Information About a Proteomics Experiment) guidelines with the advance of new technologies and techniques. Furthermore, new standards are currently either in the final stages of completion (proBed and proBAM for proteogenomics results as well as PEFF) or in early stages of design (a spectral library standard format, a universal spectrum identifier, the qcML quality control format, and the Protein Expression Interface (PROXI) web services Application Programming Interface). In this work we review the current status of all of these aspects of the PSI, describe synergies with other efforts such as the ProteomeXchange Consortium, the Human Proteome Project, and the metabolomics community, and provide a look at future directions of the PSI.

Analysis of the outer membrane proteome and secretome of Bacteroides fragilis reveals a multiplicity of secretion mechanisms.

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Marlena M Wilson

Full Text Available Bacteroides fragilis is a widely distributed member of the human gut microbiome and an opportunistic pathogen. Cell surface molecules produced by this organism likely play important roles in colonization, communication with other microbes, and pathogenicity, but the protein composition of the outer membrane (OM and the mechanisms used to transport polypeptides into the extracellular space are poorly characterized. Here we used LC-MS/MS to analyze the OM proteome and secretome of B. fragilis NCTC 9343 grown under laboratory conditions. Of the 229 OM proteins that we identified, 108 are predicted to be lipoproteins, and 61 are predicted to be TonB-dependent transporters. Based on their proximity to genes encoding TonB-dependent transporters, many of the lipoprotein genes likely encode proteins involved in nutrient or small molecule uptake. Interestingly, protease accessibility and biotinylation experiments indicated that an unusually large fraction of the lipoproteins are cell-surface exposed. We also identified three proteins that are members of a novel family of autotransporters, multiple potential type I protein secretion systems, and proteins that appear to be components of a type VI secretion apparatus. The secretome consisted of lipoproteins and other proteins that might be substrates of the putative type I or type VI secretion systems. Our proteomic studies show that B. fragilis differs considerably from well-studied Gram-negative bacteria such as Escherichia coli in both the spectrum of OM proteins that it produces and the range of secretion strategies that it utilizes.

Global Transcriptomic and Proteomic Responses of Dehalococcoides ethenogenes Strain 195 to Fixed Nitrogen Limitation

Energy Technology Data Exchange (ETDEWEB)

Lee, Patrick K. H. [University of California, Berkeley; Dill, Brian [ORNL; Louie, Tiffany S. [University of California, Berkeley; Shah, Manesh B [ORNL; Verberkmoes, Nathan C [ORNL; Andersen, Gary L. [Lawrence Berkeley National Laboratory (LBNL); Zinder, Stephen H. [Cornell University; Alvarez-Cohen, Lisa [Lawrence Berkeley National Laboratory (LBNL)

2012-01-01

Bacteria of the genus Dehalococcoides play an important role in the reductive dechlorination of chlorinated ethenes. A systems level approach was taken in this study to examine the global transcriptomic and proteomic responses of exponentially growing D. ethenogenes strain 195 to fixed nitrogen limitation (FNL) as dechlorination activity and cell yield both decrease during FNL. As expected, the nitrogen-fixing (nif) genes were differentially up-regulated in the transcriptome and proteome of strain 195 during FNL. Aside from the nif operon, a putative methylglyoxal synthase-encoding gene (DET1576), the product of which is predicted to catalyze the formation of the toxic electrophile methylglyoxal and implicated in the uncoupling of anabolism from catabolism in bacteria, was strongly up-regulated in the transcriptome and could potentially play a role in the observed growth inhibition during FNL. Carbon catabolism genes were generally down regulated in response to FNL and a number of transporters were differentially regulated in response to nitrogen limitation, with some playing apparent roles in nitrogen acquisition while others were associated with general stress responses. A number of genes related to the functions of nucleotide synthesis, replication, transcription, translation, and post-translational modifications were also differentially expressed. One gene coding for a putative reductive dehalogenase (DET1545) and a number coding for oxidoreductases, which have implications in energy generation and redox reactions, were also differentially regulated. Interestingly, most of the genes within the multiple integrated elements were not differentially expressed. Overall, this study elucidates the molecular responses of strain 195 to FNL and identifies differentially expressed genes that are potential biomarkers to evaluate environmental cellular nitrogen status.

Transcriptome of extracellular vesicles released by hepatocytes.

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Felix Royo

Full Text Available The discovery that the cells communicate through emission of vesicles has opened new opportunities for better understanding of physiological and pathological mechanisms. This discovery also provides a novel source for non-invasive disease biomarker research. Our group has previously reported that hepatocytes release extracellular vesicles with protein content reflecting the cell-type of origin. Here, we show that the extracellular vesicles released by hepatocytes also carry RNA. We report the messenger RNA composition of extracellular vesicles released in two non-tumoral hepatic models: primary culture of rat hepatocytes and a progenitor cell line obtained from a mouse foetal liver. We describe different subpopulations of extracellular vesicles with different densities and protein and RNA content. We also show that the RNA cargo of extracellular vesicles released by primary hepatocytes can be transferred to rat liver stellate-like cells and promote their activation. Finally, we provide in vitro and in vivo evidence that liver-damaging drugs galactosamine, acetaminophen, and diclofenac modify the RNA content of these vesicles. To summarize, we show that the extracellular vesicles secreted by hepatocytes contain various RNAs. These vesicles, likely to be involved in the activation of stellate cells, might become a new source for non-invasive identification of the liver toxicity markers.

A high-quality catalog of the Drosophila melanogaster proteome

DEFF Research Database (Denmark)

Brunner, Erich; Ahrens, Christian H.; Mohanty, Sonaly

2007-01-01

% of the predicted Drosophila melanogaster proteome by detecting 9,124 proteins from 498,000 redundant and 72,281 distinct peptide identifications. This unprecedented high proteome coverage for a complex eukaryote was achieved by combining sample diversity, multidimensional biochemical fractionation and analysis...

Effect of Urea and Thiourea on Generation of Xenogeneic Extracellular Matrix Scaffolds for Tissue Engineering

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Wong, Maelene L.; Wong, Janelle L.; Horn, Rebecca M.; Sannajust, Kimberley C.; Rice, Dawn A.

2016-01-01

Effective solubilization of proteins by chaotropes in proteomic applications motivates their use in solubilization-based antigen removal/decellularization strategies. A high urea concentration has previously been reported to significantly reduce lipophilic antigen content of bovine pericardium (BP); however, structure and function of the resultant extracellular matrix (ECM) scaffold were compromised. It has been recently demonstrated that in vivo ECM scaffold fate is determined by two primary outcome measures as follows: (1) sufficient reduction in antigen content to avoid graft-specific adaptive immune responses and (2) maintenance of native ECM structural proteins to avoid graft-specific innate responses. In this work, we assessed residual antigenicity, ECM architecture, ECM content, thermal stability, and tensile properties of BP subjected to a gradient of urea concentrations to determine whether an intermediate concentration exists at which both antigenicity and structure–function primary outcome measures for successful in vivo scaffold outcome can simultaneously be achieved. Alteration in tissue structure–function properties at various urea concentrations with decreased effectiveness for antigen removal makes use of urea-mediated antigen removal unlikely to be suitable for functional scaffold generation. PMID:27230226

Regional differences of the urinary proteomes in healthy Chinese individuals

OpenAIRE

Qin, Weiwei; Wu, Jianqiang; Pan, Li; Zhang, Fanshuang; Wang, Xiaorong; Zhang, Biao; Shan, Guangliang; Gao, Youhe

2017-01-01

Urine is a promising biomarker source for clinical proteomics studies. Although regional physiological differences are common in multi-center clinical studies, the presence of significant differences in the urinary proteomes of individuals from different regions remains unknown. In this study, morning urine samples were collected from healthy urban residents in three regions of China and urinary proteins were preserved using a membrane-based method (Urimem). The urine proteomes of 27 normal s...

Proteomic analyses of host and pathogen responses during bovine mastitis.

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Boehmer, Jamie L

2011-12-01

The pursuit of biomarkers for use as clinical screening tools, measures for early detection, disease monitoring, and as a means for assessing therapeutic responses has steadily evolved in human and veterinary medicine over the past two decades. Concurrently, advances in mass spectrometry have markedly expanded proteomic capabilities for biomarker discovery. While initial mass spectrometric biomarker discovery endeavors focused primarily on the detection of modulated proteins in human tissues and fluids, recent efforts have shifted to include proteomic analyses of biological samples from food animal species. Mastitis continues to garner attention in veterinary research due mainly to affiliated financial losses and food safety concerns over antimicrobial use, but also because there are only a limited number of efficacious mastitis treatment options. Accordingly, comparative proteomic analyses of bovine milk have emerged in recent years. Efforts to prevent agricultural-related food-borne illness have likewise fueled an interest in the proteomic evaluation of several prominent strains of bacteria, including common mastitis pathogens. The interest in establishing biomarkers of the host and pathogen responses during bovine mastitis stems largely from the need to better characterize mechanisms of the disease, to identify reliable biomarkers for use as measures of early detection and drug efficacy, and to uncover potentially novel targets for the development of alternative therapeutics. The following review focuses primarily on comparative proteomic analyses conducted on healthy versus mastitic bovine milk. However, a comparison of the host defense proteome of human and bovine milk and the proteomic analysis of common veterinary pathogens are likewise introduced.

When proteomics reveals unsuspected roles: the plastoglobule example

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Claire eBréhélin

2013-04-01

Full Text Available Plastoglobules are globular compartments found in plastids. Before initial proteomic studies were published, these particles were often viewed as passive lipid droplets whose unique role was to store lipids coming from the thylakoid turn-over, or to accumulate carotenoids in the chromoplasts. Yet, two proteomic studies, published concomitantly, suggested for the first time that plastoglobules are more than "junk cupboards" for lipids. Indeed, both studies demonstrated that plastoglobules do not only include structural proteins belonging to the plastoglobulin / fibrillin family, but also contain active enzymes. The specific plastoglobule localization of these enzymes has been confirmed by different approaches such as immunogold localization and GFP protein fusions, thus providing evidence that plastoglobules actively participate in diverse pathways of plastid metabolism. These proteomic studies have been the basis for numerous recent works investigating plastoglobule function. However, a lot still needs to be discovered about the molecular composition and the role of plastoglobules. In this chapter, we will describe how the proteomic approaches have launched new perspectives on plastoglobule functions.

Plant plasma membrane proteomics for improving cold tolerance

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Daisuke eTakahashi

2013-04-01

Full Text Available Plants are always exposed to various stresses. We have focused on freezing stress, which causes serious problems for agricultural management. When plants suffer freeze-induced damage, the plasma membrane is thought to be the primary site of injury because of its central role in regulation of various cellular processes. Cold tolerant species, however, adapt to such freezing conditions by modifying cellular components and functions (cold acclimation. One of the most important adaptation mechanisms to freezing is alteration of plasma membrane compositions and functions. Advanced proteomic technologies have succeeded in identification of many candidates that may play roles in adaptation of the plasma membrane to freezing stress. Proteomics results suggest that adaptations of plasma membrane functions to low temperature are associated with alterations of protein compositions during cold acclimation. Some of proteins identified by proteomic approaches have been verified their functional roles in freezing tolerance mechanisms further. Thus, accumulation of proteomic results in the plasma membrane is of importance for application to molecular breeding efforts to increase cold tolerance in crops.

Transcription factor σB plays an important role in the production of extracellular membrane-derived vesicles in Listeria monocytogenes.

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Jung Hwa Lee

Full Text Available Gram-negative bacteria produce extracellular outer membrane vesicles (OMVs that interact with host cells. Unlike Gram-negative bacteria, less is known about the production and role of extracellular membrane vesicles (MVs in Gram-positive bacteria. The food-borne pathogen Listeria monocytogenes can survive under extreme environmental and energy stress conditions and the transcription factor σ(B is involved in this survival ability. Here, we first determined the production of MVs from L. monocytogenes and evaluated whether general stress transcription factor σ(B affected production of MVs in L. monocytogenes. L. monocytogenes secreted MVs during in vitro broth culture. The wild-type strain actively produced MVs approximately nine times more and also produced more intact shapes of MVs than those of the isogenic ΔsigB mutant. A proteomic analysis showed that 130 and 89 MV proteins were identified in the wild-type and ΔsigB mutant strains, respectively. Wild-type strain-derived MVs contained proteins regulated by σ(B such as transporters (OpuCA and OpuCC, stress response (Kat, metabolism (LacD, translation (InfC, and cell division protein (FtsZ. Gene Ontology (GO enrichment analysis showed that wild-type-derived MV proteins corresponded to several GO terms, including response to stress (heat, acid, and bile resistance and extracellular polysaccharide biosynthetic process, but not the ΔsigB mutant. Internalin B (InlB was almost three times more contained in MVs derived from the wild-type strain than in MVs derived from the ΔsigB mutant. Taken together, these results suggest that σ(B plays a pivotal role in the production of MVs and protein profiles contained in MVs. L. monocytogenes MVs may contribute to host infection and survival ability under various stressful conditions.

Isolation of Extracellular Polymeric Substances from Biofilms of the Thermoacidophilic Archaeon Sulfolobus acidocaldarius.

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Jachlewski, Silke; Jachlewski, Witold D; Linne, Uwe; Bräsen, Christopher; Wingender, Jost; Siebers, Bettina

2015-01-01

Extracellular polymeric substances (EPS) are the major structural and functional components of microbial biofilms. The aim of this study was to establish a method for EPS isolation from biofilms of the thermoacidophilic archaeon, Sulfolobus acidocaldarius, as a basis for EPS analysis. Biofilms of S. acidocaldarius were cultivated on the surface of gellan gum-solidified Brock medium at 78°C for 4 days. Five EPS extraction methods were compared, including shaking of biofilm suspensions in phosphate buffer, cation-exchange resin (CER) extraction, and stirring with addition of EDTA, crown ether, or NaOH. With respect to EPS yield, impact on cell viability, and compatibility with subsequent biochemical analysis, the CER extraction method was found to be the best suited isolation procedure resulting in the detection of carbohydrates and proteins as the major constituents and DNA as a minor component of the EPS. Culturability of CER-treated cells was not impaired. Analysis of the extracellular proteome using two-dimensional gel electrophoresis resulted in the detection of several hundreds of protein spots, mainly with molecular masses of 25-116 kDa and pI values of 5-8. Identification of proteins suggested a cytoplasmic origin for many of these proteins, possibly released via membrane vesicles or biofilm-inherent cell lysis during biofilm maturation. Functional analysis of EPS proteins, using fluorogenic substrates as well as zymography, demonstrated the activity of diverse enzyme classes, such as proteases, lipases, esterases, phosphatases, and glucosidases. In conclusion, the CER extraction method, as previously applied to bacterial biofilms, also represents a suitable method for isolation of water soluble EPS from the archaeal biofilms of S. acidocaldarius, allowing the investigation of composition and function of EPS components in these types of biofilms.

Isolation of extracellular polymeric substances from biofilms of the thermoacidophilic archaeon Sulfolobus acidocaldarius

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Silke eJachlewski

2015-08-01

Full Text Available Extracellular polymeric substances (EPS are the major structural and functional components of microbial biofilms. The aim of this study was to establish a method for EPS isolation from biofilms of the thermoacidophilic archaeon Sulfolobus acidocaldarius as a basis for EPS analysis. Biofilms of S. acidocaldarius were cultivated on the surface of gellan gum-solidified Brock medium at 78 °C for 4 days. Five EPS extraction methods were compared, including shaking of biofilm suspensions in phosphate buffer, cation-exchange resin (CER extraction and stirring with addition of EDTA, crown ether or NaOH. With respect to EPS yield, impact on cell viability and compatibility with subsequent biochemical analysis, the CER extraction method was found to be the best suited isolation procedure resulting in the detection of carbohydrates and proteins as the major constituents and DNA as a minor component of the EPS. Culturability of CER-treated cells was not impaired. Analysis of the extracellular proteome using two-dimensional gel electrophoresis resulted in the detection of several hundredshundred of protein spots, mainly with molecular masses of 25 kDa to 116 kDa and pI values of 5 to 8. Identification of proteins suggested a cytoplasmic origin for many of these proteins, possibly released via membrane vesicles or biofilm-inherent cell lysis during biofilm maturation. Functional analysis of EPS proteins, using fluorogenic substrates as well as zymography, demonstrated the activity of diverse groups of enzymes such as proteases, lipases, esterases, phosphatases and glucosidases. In conclusion, the CER extraction method, as previously applied to bacterial biofilms, also represents a suitable method for isolation of water soluble EPS from the archaeal biofilms of S. acidocaldarius, allowing the investigation of composition and function of EPS components in these types of biofilms.

Differential quantitative proteomics of Porphyromonas gingivalis by linear ion trap mass spectrometry: Non-label methods comparison, q-values and LOWESS curve fitting

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Xia, Qiangwei; Wang, Tiansong; Park, Yoonsuk; Lamont, Richard J.; Hackett, Murray

2007-01-01

Differential analysis of whole cell proteomes by mass spectrometry has largely been applied using various forms of stable isotope labeling. While metabolic stable isotope labeling has been the method of choice, it is often not possible to apply such an approach. Four different label free ways of calculating expression ratios in a classic "two-state" experiment are compared: signal intensity at the peptide level, signal intensity at the protein level, spectral counting at the peptide level, and spectral counting at the protein level. The quantitative data were mined from a dataset of 1245 qualitatively identified proteins, about 56% of the protein encoding open reading frames from Porphyromonas gingivalis, a Gram-negative intracellular pathogen being studied under extracellular and intracellular conditions. Two different control populations were compared against P. gingivalis internalized within a model human target cell line. The q-value statistic, a measure of false discovery rate previously applied to transcription microarrays, was applied to proteomics data. For spectral counting, the most logically consistent estimate of random error came from applying the locally weighted scatter plot smoothing procedure (LOWESS) to the most extreme ratios generated from a control technical replicate, thus setting upper and lower bounds for the region of experimentally observed random error.

Lysophosphatidic acid receptor activation affects the C13NJ microglia cell line proteome leading to alterations in glycolysis, motility, and cytoskeletal architecture

Science.gov (United States)

Bernhart, Eva; Kollroser, Manfred; Rechberger, Gerald; Reicher, Helga; Heinemann, Akos; Schratl, Petra; Hallström, Seth; Wintersperger, Andrea; Nusshold, Christoph; DeVaney, Trevor; Zorn-Pauly, Klaus; Malli, Roland; Graier, Wolfgang; Malle, Ernst; Sattler, Wolfgang

2014-01-01

Microglia, the immunocompetent cells of the CNS, are rapidly activated in response to injury and microglia migration towards and homing at damaged tissue plays a key role in CNS regeneration. Lysophosphatidic acid (LPA) is involved in signaling events evoking microglia responses through cognate G protein-coupled receptors. Here we show that human immortalized C13NJ microglia express LPA receptor subtypes LPA1, LPA2, and LPA3 on mRNA and protein level. LPA activation of C13NJ cells induced Rho and extracellular signal-regulated kinase activation and enhanced cellular ATP production. In addition, LPA induced process retraction, cell spreading, led to pronounced changes of the actin cytoskeleton and reduced cell motility, which could be reversed by inhibition of Rho activity. To get an indication about LPA-induced global alterations in protein expression patterns a 2-D DIGE/LC-ESI-MS proteomic approach was applied. On the proteome level the most prominent changes in response to LPA were observed for glycolytic enzymes and proteins regulating cell motility and/or cytoskeletal dynamics. The present findings suggest that naturally occurring LPA is a potent regulator of microglia biology. This might be of particular relevance in the pathophysiological context of neurodegenerative disorders where LPA concentrations can be significantly elevated in the CNS. PMID:19899077

Proteome regulation during Olea europaea fruit development.

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Linda Bianco

Full Text Available Widespread in the Mediterranean basin, Olea europaea trees are gaining worldwide popularity for the nutritional and cancer-protective properties of the oil, mechanically extracted from ripe fruits. Fruit development is a physiological process with remarkable impact on the modulation of the biosynthesis of compounds affecting the quality of the drupes as well as the final composition of the olive oil. Proteomics offers the possibility to dig deeper into the major changes during fruit development, including the important phase of ripening, and to classify temporal patterns of protein accumulation occurring during these complex physiological processes.In this work, we started monitoring the proteome variations associated with olive fruit development by using comparative proteomics coupled to mass spectrometry. Proteins extracted from drupes at three different developmental stages were separated on 2-DE and subjected to image analysis. 247 protein spots were revealed as differentially accumulated. Proteins were identified from a total of 121 spots and discussed in relation to olive drupe metabolic changes occurring during fruit development. In order to evaluate if changes observed at the protein level were consistent with changes of mRNAs, proteomic data produced in the present work were compared with transcriptomic data elaborated during previous studies.This study identifies a number of proteins responsible for quality traits of cv. Coratina, with particular regard to proteins associated to the metabolism of fatty acids, phenolic and aroma compounds. Proteins involved in fruit photosynthesis have been also identified and their pivotal contribution in oleogenesis has been discussed. To date, this study represents the first characterization of the olive fruit proteome during development, providing new insights into fruit metabolism and oil accumulation process.

Proteome regulation during Olea europaea fruit development.

Science.gov (United States)

Bianco, Linda; Alagna, Fiammetta; Baldoni, Luciana; Finnie, Christine; Svensson, Birte; Perrotta, Gaetano

2013-01-01

Widespread in the Mediterranean basin, Olea europaea trees are gaining worldwide popularity for the nutritional and cancer-protective properties of the oil, mechanically extracted from ripe fruits. Fruit development is a physiological process with remarkable impact on the modulation of the biosynthesis of compounds affecting the quality of the drupes as well as the final composition of the olive oil. Proteomics offers the possibility to dig deeper into the major changes during fruit development, including the important phase of ripening, and to classify temporal patterns of protein accumulation occurring during these complex physiological processes. In this work, we started monitoring the proteome variations associated with olive fruit development by using comparative proteomics coupled to mass spectrometry. Proteins extracted from drupes at three different developmental stages were separated on 2-DE and subjected to image analysis. 247 protein spots were revealed as differentially accumulated. Proteins were identified from a total of 121 spots and discussed in relation to olive drupe metabolic changes occurring during fruit development. In order to evaluate if changes observed at the protein level were consistent with changes of mRNAs, proteomic data produced in the present work were compared with transcriptomic data elaborated during previous studies. This study identifies a number of proteins responsible for quality traits of cv. Coratina, with particular regard to proteins associated to the metabolism of fatty acids, phenolic and aroma compounds. Proteins involved in fruit photosynthesis have been also identified and their pivotal contribution in oleogenesis has been discussed. To date, this study represents the first characterization of the olive fruit proteome during development, providing new insights into fruit metabolism and oil accumulation process.

Sherlock Holmes and the proteome--a detective story.

Science.gov (United States)

Righetti, Pier Giorgio; Boschetti, Egisto

2007-02-01

The performance of a hexapeptide ligand library in capturing the 'hidden proteome' is illustrated and evaluated. This library, insolubilized on an organic polymer and available under the trade name 'Equalizer Bead Technology', acts by capturing all components of a given proteome, by concentrating rare and very rare proteins, and simultaneously diluting the abundant ones. This results in a proteome of 'normalized' relative abundances, amenable to analysis by MS and any other analytical tool. Examples are given of analysis of human urine and serum, as well as cell and tissue lysates, such as Escherichia coli and Saccharomyces cerevisiae extracts. Another important application is impurity tracking and polishing of recombinant DNA products, especially biopharmaceuticals meant for human consumption.

Systems biology integration of proteomic data in rodent models of depression reveals involvement of the immune response and glutamatergic signaling.

Science.gov (United States)

Carboni, Lucia; Nguyen, Thanh-Phuong; Caberlotto, Laura

2016-12-01

The pathophysiological basis of major depression is incompletely understood. Recently, numerous proteomic studies have been performed in rodent models of depression to investigate the molecular underpinnings of depressive-like behaviours with an unbiased approach. The objective of the study is to integrate the results of these proteomic studies in depression models to shed light on the most relevant molecular pathways involved in the disease. Network analysis is performed integrating preexisting proteomic data from rodent models of depression. The IntAct mouse and the HRPD are used as reference protein-protein interaction databases. The functionality analyses of the networks are then performed by testing overrepresented GO biological process terms and pathways. Functional enrichment analyses of the networks revealed an association with molecular processes related to depression in humans, such as those involved in the immune response. Pathways impacted by clinically effective antidepressants are modulated, including glutamatergic signaling and neurotrophic responses. Moreover, dysregulations of proteins regulating energy metabolism and circadian rhythms are implicated. The comparison with protein pathways modulated in depressive patients revealed significant overlapping. This systems biology study supports the notion that animal models can contribute to the research into the biology and therapeutics of depression. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An update on the mouse liver proteome

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Borlak Jürgen

2009-09-01

Full Text Available Abstract Background Decoding of the liver proteome is subject of intense research, but hampered by methodological constraints. We recently developed an improved protocol for studying rat liver proteins based on 2-DE-MALDI-TOF-MS peptide mass finger printing. This methodology was now applied to develop a mouse liver protein database. Results Liver proteins were extracted by two different lysis buffers in sequence followed by a liquid-phase IEF pre-fractionation and separation of proteins by 2 DE at two different pH ranges, notably 5-8 and 7-10. Based on 9600 in gel digests a total of 643 mouse liver proteins with high sequence coverage (> 20 peptides per protein could be identified by MALDI-TOF-MS peptide mass finger printing. Notably, 255 proteins are novel and have not been reported so far by conventional two-dimensional electrophoresis proteome mapping. Additionally, the results of the present findings for mouse liver were compared to published data of the rat proteome to compile as many proteins as possible in a rodent liver database. Conclusion Based on 2-DE MALDI-TOF-MS a significantly improved proteome map of mouse liver was obtained. We discuss some prominent members of newly identified proteins for a better understanding of liver biology.

Quantitative proteomic assessment of very early cellular signaling events

DEFF Research Database (Denmark)

Dengjel, Joern; Akimov, Vyacheslav; Olsen, Jesper V

2007-01-01

Technical limitations have prevented proteomic analyses of events occurring less than 30 s after signal initiation. We developed an automated, continuous quench-flow system allowing quantitative proteomic assessment of very early cellular signaling events (qPACE) with a time resolution of 1 s...

Proteomic Technologies for the Study of Osteosarcoma

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Stephanie D. Byrum

2012-01-01

Full Text Available Osteosarcoma is the most common primary bone cancer of children and is established during stages of rapid bone growth. The disease is a consequence of immature osteoblast differentiation, which gives way to a rapidly synthesized incompletely mineralized and disorganized bone matrix. The mechanism of osteosarcoma tumorogenesis is poorly understood, and few proteomic studies have been used to interrogate the disease thus far. Accordingly, these studies have identified proteins that have been known to be associated with other malignancies, rather than being osteosarcoma specific. In this paper, we focus on the growing list of available state-of-the-art proteomic technologies and their specific application to the discovery of novel osteosarcoma diagnostic and therapeutic targets. The current signaling markers/pathways associated with primary and metastatic osteosarcoma that have been identified by early-stage proteomic technologies thus far are also described.

The Use of Proteomics in Assisted Reproduction.

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Kosteria, Ioanna; Anagnostopoulos, Athanasios K; Kanaka-Gantenbein, Christina; Chrousos, George P; Tsangaris, George T

2017-01-01

Despite the explosive increase in the use of Assisted Reproductive Technologies (ART) over the last 30 years, their success rates remain suboptimal. Proteomics is a rapidly-evolving technology-driven science that has already been widely applied in the exploration of human reproduction and fertility, providing useful insights into its physiology and leading to the identification of numerous proteins that may be potential biomarkers and/or treatment targets of a successful ART pregnancy. Here we present a brief overview of the techniques used in proteomic analyses and attempt a comprehensive presentation of recent data from mass spectrometry-based proteomic studies in humans, regarding all components of ARTs, including the male and female gamete, the derived zygote and embryo, the endometrium and, finally, the ART offspring both pre- and postnatally. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Proteomic analysis of minute amount of colonic biopsies by enteroscopy sampling

Energy Technology Data Exchange (ETDEWEB)

Liu, Xing [Department of Analytical Chemistry and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (China); Xu, Yanli [Fuyang People’s Hospital (China); Meng, Qian [Department of Analytical Chemistry and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (China); Zheng, Qingqing [Digestive Endoscopic Center, Shanghai Jiaotong University Affiliated Sixth People’s Hospital (China); Wu, Jianhong [Department of Analytical Chemistry and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (China); Wang, Chen; Jia, Weiping [Shanghai Key Laboratory of Diabetes Mellitus, Department of Endocrinology and Metabolism, Shanghai Diabetes Institute, Shanghai Clinical Center for Diabetes, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital (China); Figeys, Daniel [Department of Biochemistry, Microbiology and Immunology, and Department of Chemistry and Biomolecular Sciences, University of Ottawa (Canada); Chang, Ying, E-mail: emulan@163.com [Digestive Endoscopic Center, Shanghai Jiaotong University Affiliated Sixth People’s Hospital (China); Zhou, Hu, E-mail: zhouhu@simm.ac.cn [Department of Analytical Chemistry and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (China)

2016-08-05

Colorectal cancer (CRC) is one of the most common types of malignant tumor worldwide. Currently, although many researchers have been devoting themselves in CRC studies, the process of locating biomarkers for CRC early diagnosis and prognostic is still very slow. Using a centrifugal proteomic reactor-based proteomic analysis of minute amount of colonic biopsies by enteroscopy sampling, 2620 protein groups were quantified between cancer mucosa and adjacent normal colorectal mucosa. Of which, 403 protein groups were differentially expressed with statistic significance between cancer and normal tissues, including 195 up-regulated and 208 down-regulated proteins in cancer tissues. Three proteins (SOD3, PRELP and NGAL) were selected for further Western blot validation. And the resulting Western blot experimental results were consistent with the quantitative proteomic data. SOD3 and PRELP are down-regulated in CRC mucosa comparing to adjacent normal tissue, while NGAL is up-regulated in CRC mucosa. In conclusion, the centrifugal proteomic reactor-based label-free quantitative proteomic approach provides a highly sensitive and powerful tool for analyzing minute protein sample from tiny colorectal biopsies, which may facilitate CRC biomarkers discovery for diagnoses and prognoses. -- Highlights: •Minute amount of colonic biopsies by endoscopy is suitable for proteomic analysis. •Centrifugal proteomic reactor can be used for processing tiny clinic biopsy sample. •SOD3 and PRELP are down-regulated in CRC, while NGAL is up-regulated in CRC.

Proteomic analysis of minute amount of colonic biopsies by enteroscopy sampling

International Nuclear Information System (INIS)

Liu, Xing; Xu, Yanli; Meng, Qian; Zheng, Qingqing; Wu, Jianhong; Wang, Chen; Jia, Weiping; Figeys, Daniel; Chang, Ying; Zhou, Hu

2016-01-01

Colorectal cancer (CRC) is one of the most common types of malignant tumor worldwide. Currently, although many researchers have been devoting themselves in CRC studies, the process of locating biomarkers for CRC early diagnosis and prognostic is still very slow. Using a centrifugal proteomic reactor-based proteomic analysis of minute amount of colonic biopsies by enteroscopy sampling, 2620 protein groups were quantified between cancer mucosa and adjacent normal colorectal mucosa. Of which, 403 protein groups were differentially expressed with statistic significance between cancer and normal tissues, including 195 up-regulated and 208 down-regulated proteins in cancer tissues. Three proteins (SOD3, PRELP and NGAL) were selected for further Western blot validation. And the resulting Western blot experimental results were consistent with the quantitative proteomic data. SOD3 and PRELP are down-regulated in CRC mucosa comparing to adjacent normal tissue, while NGAL is up-regulated in CRC mucosa. In conclusion, the centrifugal proteomic reactor-based label-free quantitative proteomic approach provides a highly sensitive and powerful tool for analyzing minute protein sample from tiny colorectal biopsies, which may facilitate CRC biomarkers discovery for diagnoses and prognoses. -- Highlights: •Minute amount of colonic biopsies by endoscopy is suitable for proteomic analysis. •Centrifugal proteomic reactor can be used for processing tiny clinic biopsy sample. •SOD3 and PRELP are down-regulated in CRC, while NGAL is up-regulated in CRC.

PRIDE Inspector Toolsuite: Moving Toward a Universal Visualization Tool for Proteomics Data Standard Formats and Quality Assessment of ProteomeXchange Datasets.

Science.gov (United States)

Perez-Riverol, Yasset; Xu, Qing-Wei; Wang, Rui; Uszkoreit, Julian; Griss, Johannes; Sanchez, Aniel; Reisinger, Florian; Csordas, Attila; Ternent, Tobias; Del-Toro, Noemi; Dianes, Jose A; Eisenacher, Martin; Hermjakob, Henning; Vizcaíno, Juan Antonio

2016-01-01

The original PRIDE Inspector tool was developed as an open source standalone tool to enable the visualization and validation of mass-spectrometry (MS)-based proteomics data before data submission or already publicly available in the Proteomics Identifications (PRIDE) database. The initial implementation of the tool focused on visualizing PRIDE data by supporting the PRIDE XML format and a direct access to private (password protected) and public experiments in PRIDE.The ProteomeXchange (PX) Consortium has been set up to enable a better integration of existing public proteomics repositories, maximizing its benefit to the scientific community through the implementation of standard submission and dissemination pipelines. Within the Consortium, PRIDE is focused on supporting submissions of tandem MS data. The increasing use and popularity of the new Proteomics Standards Initiative (PSI) data standards such as mzIdentML and mzTab, and the diversity of workflows supported by the PX resources, prompted us to design and implement a new suite of algorithms and libraries that would build upon the success of the original PRIDE Inspector and would enable users to visualize and validate PX "complete" submissions. The PRIDE Inspector Toolsuite supports the handling and visualization of different experimental output files, ranging from spectra (mzML, mzXML, and the most popular peak lists formats) and peptide and protein identification results (mzIdentML, PRIDE XML, mzTab) to quantification data (mzTab, PRIDE XML), using a modular and extensible set of open-source, cross-platform libraries. We believe that the PRIDE Inspector Toolsuite represents a milestone in the visualization and quality assessment of proteomics data. It is freely available at http://github.com/PRIDE-Toolsuite/. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Application of proteomics to investigate barley-Fusarium graminearum interaction

DEFF Research Database (Denmark)

Yang, Fen

in plants under low N and iv) proteomes of uninfected plants were similar under two N levels. Correlation of level of proteolysis induced by the fungus with measurement of Fusarium-damaged kernels, fungal biomass and mycotoxin levels indicated that FHB was more severe in barley with low N. In Chapter 3......, the molecular mechanisms of barley defense to Fusarium graminearum at the early infection stage were studied. Antibodies against barley β-amylases were shown to be the markers for infection at proteome level and for selection of the time for proteome analysis before extensive degradation caused by the fungus...... the disease. Due to the advantages of gel-based proteomics that differentially expressed proteins involved in the interaction can be directly detected by comparing protein profiles displayed on 2-D gels, it is used as a tool for studying the barley- Fusarium graminearum interaction form three different...

Detergents: Friends not foes for high-performance membrane proteomics toward precision medicine.

Science.gov (United States)

Zhang, Xi

2017-02-01

Precision medicine, particularly therapeutics, emphasizes the atomic-precise, dynamic, and systems visualization of human membrane proteins and their endogenous modifiers. For years, bottom-up proteomics has grappled with removing and avoiding detergents, yet faltered at the therapeutic-pivotal membrane proteins, which have been tackled by classical approaches and are known for decades refractory to single-phase aqueous or organic denaturants. Hydrophobicity and aggregation commonly challenge tissue and cell lysates, biofluids, and enriched samples. Frequently, expected membrane proteins and peptides are not identified by shotgun bottom-up proteomics, let alone robust quantitation. This review argues the cause of this proteomic crisis is not detergents per se, but the choice of detergents. Recently, inclusion of compatible detergents for membrane protein extraction and digestion has revealed stark improvements in both quantitative and structural proteomics. This review analyzes detergent properties behind recent proteomic advances, and proposes that rational use of detergents may reconcile outstanding membrane proteomics dilemmas, enabling ultradeep coverage and minimal artifacts for robust protein and endogenous PTM measurements. The simplicity of detergent tools confers bottom-up membrane proteomics the sophistication toward precision medicine. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tissue localization and extracellular matrix degradation by PI, PII and PIII snake venom metalloproteinases: clues on the mechanisms of venom-induced hemorrhage.

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Cristina Herrera

2015-04-01

Full Text Available Snake venom hemorrhagic metalloproteinases (SVMPs of the PI, PII and PIII classes were compared in terms of tissue localization and their ability to hydrolyze basement membrane components in vivo, as well as by a proteomics analysis of exudates collected in tissue injected with these enzymes. Immunohistochemical analyses of co-localization of these SVMPs with type IV collagen revealed that PII and PIII enzymes co-localized with type IV collagen in capillaries, arterioles and post-capillary venules to a higher extent than PI SVMP, which showed a more widespread distribution in the tissue. The patterns of hydrolysis by these three SVMPs of laminin, type VI collagen and nidogen in vivo greatly differ, whereas the three enzymes showed a similar pattern of degradation of type IV collagen, supporting the concept that hydrolysis of this component is critical for the destabilization of microvessel structure leading to hemorrhage. Proteomic analysis of wound exudate revealed similarities and differences between the action of the three SVMPs. Higher extent of proteolysis was observed for the PI enzyme regarding several extracellular matrix components and fibrinogen, whereas exudates from mice injected with PII and PIII SVMPs had higher amounts of some intracellular proteins. Our results provide novel clues for understanding the mechanisms by which SVMPs induce damage to the microvasculature and generate hemorrhage.

Urine Proteomics Revealed a Significant Correlation Between Urine-Fibronectin Abundance and Estimated-GFR Decline in Patients with Bardet-Biedl Syndrome

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Marianna Caterino

2018-03-01

Full Text Available Background:/Aims: Renal disease is a common cause of morbidity in patients with Bardet-Biedl syndrome (BBS, however the severity of kidney dysfunction is highly variable. To date, there is little information on the pathogenesis, the risk and predictor factors for poor renal outcome in this setting. The present study aims to analyze the spectrum of urinary proteins in BBS patients, in order to potentially identify 1 disease-specific proteomic profiles that may differentiate the patients from normal subjects; 2 urinary markers of renal dysfunction. Methods: Fourteen individuals (7 males and 7 females with a clinical diagnosis of BBS have been selected in this study. A pool of 10 aged-matched males and 10 aged-matched females have been used as controls for proteomic analysis. The glomerular filtration rate (eGFR has been estimated using the CKD-EPI formula. Variability of eGFR has been retrospectively assessed calculating average annual eGFR decline (ΔeGFR in a mean follow-up period of 4 years (3-7. Results: 42 proteins were significantly over- or under-represented in BBS patients compared with controls; the majority of these proteins are involved in fibrosis, cell adhesion and extracellular matrix organization. Statistic studies revealed a significant correlation between urine fibronectin (u-FN (r2=0.28; p<0.05, CD44 antigen (r2 =0.35; p<0.03 and lysosomal alfa glucosidase ( r20.27; p<0.05 abundance with the eGFR. In addition, u-FN (r2 =0.2389; p<0.05 was significantly correlated with ΔeGFR. Conclusion: The present study demonstrates that urine proteome of BBS patients differs from that of normal subjects; in addition, kidney dysfunction correlated with urine abundance of known markers of renal fibrosis.

Data from proteome analysis of Lasiodiplodia theobromae (Botryosphaeriaceae

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Carla C. Uranga

2017-08-01

Full Text Available Trunk disease fungi are a global problem affecting many economically important fruiting trees. The Botryosphaeriaceae are a family of trunk disease fungi that require detailed biochemical characterization in order to gain insight into their pathogenicity. The application of a modified Folch extraction to protein extraction from the Botryosphaeriaceae Lasiodiplodia theobromae generated an unprecedented data set of protein identifications from fragmentation analysis and de novo peptide sequencing of its proteome. This article contains data from protein identifications obtained from a database-dependent fragmentation analysis using three different proteomics algorithms (MSGF, Comet and X! Tandem via the SearchGUI proteomics pipeline program and de novo peptide sequencing. Included are data sets of gene ontology annotations using an all-Uniprot ontology database, as well as a Saccharomyces cerevisiae-only and a Candida albicans-only ontology database, in order to discern between those proteins involved in common functions with S. cerevisiae and those in common with the pathogenic yeast C. albicans. Our results reveal the proteome of L. theobromae contains more ontological categories in common to C. albicans, yet possesses a much wider metabolic repertoire than any of the yeasts studied in this work. Many novel proteins of interest were identified for further biochemical characterization and annotation efforts, as further discussed in the article referencing this article (1. Interactive Cytoscape networks of molecular functions of identified peptides using an all-Uniprot ontological database are included. Data, including raw data, are available via ProteomeXchange with identifier PXD005283.

Proteomics in the investigation of HIV-1 interactions with host proteins.

Science.gov (United States)

Li, Ming

2015-02-01

Productive HIV-1 infection depends on host machinery, including a broad array of cellular proteins. Proteomics has played a significant role in the discovery of HIV-1 host proteins. In this review, after a brief survey of the HIV-1 host proteins that were discovered by proteomic analyses, I focus on analyzing the interactions between the virion and host proteins, as well as the technologies and strategies used in those proteomic studies. With the help of proteomics, the identification and characterization of HIV-1 host proteins can be translated into novel antiretroviral therapeutics. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteomic differences between native and tissue‐engineered tendon and ligament

Science.gov (United States)

Tew, Simon R.; Peffers, Mandy; Canty‐Laird, Elizabeth G.; Comerford, Eithne

2016-01-01

Tendons and ligaments (T/Ls) play key roles in the musculoskeletal system, but they are susceptible to traumatic or age‐related rupture, leading to severe morbidity as well as increased susceptibility to degenerative joint diseases such as osteoarthritis. Tissue engineering represents an attractive therapeutic approach to treating T/L injury but it is hampered by our poor understanding of the defining characteristics of the two tissues. The present study aimed to determine differences in the proteomic profile between native T/Ls and tissue engineered (TE) T/L constructs. The canine long digital extensor tendon and anterior cruciate ligament were analyzed along with 3D TE fibrin‐based constructs created from their cells. Native tendon and ligament differed in their content of key structural proteins, with the ligament being more abundant in fibrocartilaginous proteins. 3D T/L TE constructs contained less extracellular matrix (ECM) proteins and had a greater proportion of cellular‐associated proteins than native tissue, corresponding to their low collagen and high DNA content. Constructs were able to recapitulate native T/L tissue characteristics particularly with regard to ECM proteins. However, 3D T/L TE constructs had similar ECM and cellular protein compositions indicating that cell source may not be an important factor for T/L tissue engineering. PMID:27080496

Proteomic Investigations into Hemodialysis Therapy

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Mario Bonomini

2015-12-01

Full Text Available The retention of a number of solutes that may cause adverse biochemical/biological effects, called uremic toxins, characterizes uremic syndrome. Uremia therapy is based on renal replacement therapy, hemodialysis being the most commonly used modality. The membrane contained in the hemodialyzer represents the ultimate determinant of the success and quality of hemodialysis therapy. Membrane’s performance can be evaluated in terms of removal efficiency for unwanted solutes and excess fluid, and minimization of negative interactions between the membrane material and blood components that define the membrane’s bio(incompatibility. Given the high concentration of plasma proteins and the complexity of structural functional relationships of this class of molecules, the performance of a membrane is highly influenced by its interaction with the plasma protein repertoire. Proteomic investigations have been increasingly applied to describe the protein uremic milieu, to compare the blood purification efficiency of different dialyzer membranes or different extracorporeal techniques, and to evaluate the adsorption of plasma proteins onto hemodialysis membranes. In this article, we aim to highlight investigations in the hemodialysis setting making use of recent developments in proteomic technologies. Examples are presented of why proteomics may be helpful to nephrology and may possibly affect future directions in renal research.

Proteomic Investigations into Hemodialysis Therapy

Science.gov (United States)

Bonomini, Mario; Sirolli, Vittorio; Pieroni, Luisa; Felaco, Paolo; Amoroso, Luigi; Urbani, Andrea

2015-01-01

The retention of a number of solutes that may cause adverse biochemical/biological effects, called uremic toxins, characterizes uremic syndrome. Uremia therapy is based on renal replacement therapy, hemodialysis being the most commonly used modality. The membrane contained in the hemodialyzer represents the ultimate determinant of the success and quality of hemodialysis therapy. Membrane’s performance can be evaluated in terms of removal efficiency for unwanted solutes and excess fluid, and minimization of negative interactions between the membrane material and blood components that define the membrane’s bio(in)compatibility. Given the high concentration of plasma proteins and the complexity of structural functional relationships of this class of molecules, the performance of a membrane is highly influenced by its interaction with the plasma protein repertoire. Proteomic investigations have been increasingly applied to describe the protein uremic milieu, to compare the blood purification efficiency of different dialyzer membranes or different extracorporeal techniques, and to evaluate the adsorption of plasma proteins onto hemodialysis membranes. In this article, we aim to highlight investigations in the hemodialysis setting making use of recent developments in proteomic technologies. Examples are presented of why proteomics may be helpful to nephrology and may possibly affect future directions in renal research. PMID:26690416

Proteomic profile of acute myeloid leukaemia: A review update

African Journals Online (AJOL)

attention to the progress and advancements in cancer proteomics technology with the aim of simplifying ... hematopoietic cells leading to distinct differences ... procedures like bone marrow and tissue biopsies. [7,8]. .... patients who were subjected to transplantation ..... Boyd RS, Dyer MJ, Cain K. Proteomic analysis of b-cell.

A community proposal to integrate proteomics activities in ELIXIR

NARCIS (Netherlands)

Vizcaíno, Juan Antonio; Walzer, Mathias; Jiménez, Rafael C; Bittremieux, Wout; Bouyssié, David; Carapito, Christine; Corrales, Fernando; Ferro, Myriam; Heck, Albert J R; Horvatovich, Peter; Hubalek, Martin; Lane, Lydie; Laukens, Kris; Levander, Fredrik; Lisacek, Frederique; Novak, Petr; Palmblad, Magnus; Piovesan, Damiano; Pühler, Alfred; Schwämmle, Veit; Valkenborg, Dirk; van Rijswijk, Merlijn; Vondrasek, Jiri; Eisenacher, Martin; Martens, Lennart; Kohlbacher, Oliver

2017-01-01

Computational approaches have been major drivers behind the progress of proteomics in recent years. The aim of this white paper is to provide a framework for integrating computational proteomics into ELIXIR in the near future, and thus to broaden the portfolio of omics technologies supported by this

Asymmetric proteome equalization of the skeletal muscle proteome using a combinatorial hexapeptide library.

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Jenny Rivers

Full Text Available Immobilized combinatorial peptide libraries have been advocated as a strategy for equalization of the dynamic range of a typical proteome. The technology has been applied predominantly to blood plasma and other biological fluids such as urine, but has not been used extensively to address the issue of dynamic range in tissue samples. Here, we have applied the combinatorial library approach to the equalization of a tissue where there is also a dramatic asymmetry in the range of abundances of proteins; namely, the soluble fraction of skeletal muscle. We have applied QconCAT and label-free methodology to the quantification of the proteins that bind to the beads as the loading is progressively increased. Although some equalization is achieved, and the most abundant proteins no longer dominate the proteome analysis, at high protein loadings a new asymmetry of protein expression is reached, consistent with the formation of complex assembles of heat shock proteins, cytoskeletal elements and other proteins on the beads. Loading at different ionic strength values leads to capture of different subpopulations of proteins, but does not completely eliminate the bias in protein accumulation. These assemblies may impair the broader utility of combinatorial library approaches to the equalization of tissue proteomes. However, the asymmetry in equalization is manifest at either low and high ionic strength values but manipulation of the solvent conditions may extend the capacity of the method.

Extracellular pH Modulates Neuroendocrine Prostate Cancer Cell Metabolism and Susceptibility to the Mitochondrial Inhibitor Niclosamide

Science.gov (United States)

Ippolito, Joseph E.; Brandenburg, Matthew W.; Ge, Xia; Crowley, Jan R.; Kirmess, Kristopher M.; Som, Avik; D’Avignon, D. Andre; Arbeit, Jeffrey M.; Achilefu, Samuel; Yarasheski, Kevin E.; Milbrandt, Jeffrey

2016-01-01

Neuroendocrine prostate cancer is a lethal variant of prostate cancer that is associated with castrate-resistant growth, metastasis, and mortality. The tumor environment of neuroendocrine prostate cancer is heterogeneous and characterized by hypoxia, necrosis, and numerous mitoses. Although acidic extracellular pH has been implicated in aggressive cancer features including metastasis and therapeutic resistance, its role in neuroendocrine prostate cancer physiology and metabolism has not yet been explored. We used the well-characterized PNEC cell line as a model to establish the effects of extracellular pH (pH 6.5, 7.4, and 8.5) on neuroendocrine prostate cancer cell metabolism. We discovered that alkalinization of extracellular pH converted cellular metabolism to a nutrient consumption-dependent state that was susceptible to glucose deprivation, glutamine deprivation, and 2-deoxyglucose (2-DG) mediated inhibition of glycolysis. Conversely, acidic pH shifted cellular metabolism toward an oxidative phosphorylation (OXPHOS)-dependent state that was susceptible to OXPHOS inhibition. Based upon this mechanistic knowledge of pH-dependent metabolism, we identified that the FDA-approved anti-helminthic niclosamide depolarized mitochondrial potential and depleted ATP levels in PNEC cells whose effects were enhanced in acidic pH. To further establish relevance of these findings, we tested the effects of extracellular pH on susceptibility to nutrient deprivation and OXPHOS inhibition in a cohort of castrate-resistant prostate cancer cell lines C4-2B, PC-3, and PC-3M. We discovered similar pH-dependent toxicity profiles among all cell lines with these treatments. These findings underscore a potential importance to acidic extracellular pH in the modulation of cell metabolism in tumors and development of an emerging paradigm that exploits the synergy of environment and therapeutic efficacy in cancer. PMID:27438712