Genome-wide screening and identification of antigens for rickettsial vaccine development

USDA-ARS?s Scientific Manuscript database

The capacity to identify immunogens for vaccine development by genome-wide screening has been markedly enhanced by the availability of complete microbial genome sequences coupled to rapid proteomic and bioinformatic analysis. Critical to this genome-wide screening is in vivo testing in the context o...

Large-Scale Interaction Profiling of Protein Domains Through Proteomic Peptide-Phage Display Using Custom Peptidomes.

PubMed

Seo, Moon-Hyeong; Nim, Satra; Jeon, Jouhyun; Kim, Philip M

2017-01-01

Protein-protein interactions are essential to cellular functions and signaling pathways. We recently combined bioinformatics and custom oligonucleotide arrays to construct custom-made peptide-phage libraries for screening peptide-protein interactions, an approach we call proteomic peptide-phage display (ProP-PD). In this chapter, we describe protocols for phage display for the identification of natural peptide binders for a given protein. We finally describe deep sequencing for the analysis of the proteomic peptide-phage display.

Drug target identification in protozoan parasites

PubMed Central

Müller, Joachim; Hemphill, Andrew

2016-01-01

Introduction Despite the fact that diseases caused by protozoan parasites represent serious challenges for public health, animal production and welfare, only a limited panel of drugs has been marketed for clinical applications. Areas covered Herein, the authors investigate two strategies, namely whole organism screening and target-based drug design. The present pharmacopoeia has resulted from whole organism screening, and the mode of action and targets of selected drugs are discussed. However, the more recent extensive genome sequencing efforts and the development of dry and wet lab genomics and proteomics that allow high-throughput screening of interactions between micromolecules and recombinant proteins has resulted in target-based drug design as the predominant focus in anti-parasitic drug development. Selected examples of target-based drug design studies are presented, and calcium-dependent protein kinases, important drug targets in apicomplexan parasites, are discussed in more detail. Expert opinion Despite the enormous efforts in target-based drug development, this approach has not yet generated market-ready antiprotozoal drugs. However, whole-organism screening approaches, comprising of both in vitro and in vivo investigations, should not be disregarded. The repurposing of already approved and marketed drugs could be a suitable strategy to avoid fastidious approval procedures, especially in the case of neglected or veterinary parasitoses. PMID:27238605

Interlaboratory Study Characterizing a Yeast Performance Standard for Benchmarking LC-MS Platform Performance*

PubMed Central

Paulovich, Amanda G.; Billheimer, Dean; Ham, Amy-Joan L.; Vega-Montoto, Lorenzo; Rudnick, Paul A.; Tabb, David L.; Wang, Pei; Blackman, Ronald K.; Bunk, David M.; Cardasis, Helene L.; Clauser, Karl R.; Kinsinger, Christopher R.; Schilling, Birgit; Tegeler, Tony J.; Variyath, Asokan Mulayath; Wang, Mu; Whiteaker, Jeffrey R.; Zimmerman, Lisa J.; Fenyo, David; Carr, Steven A.; Fisher, Susan J.; Gibson, Bradford W.; Mesri, Mehdi; Neubert, Thomas A.; Regnier, Fred E.; Rodriguez, Henry; Spiegelman, Cliff; Stein, Stephen E.; Tempst, Paul; Liebler, Daniel C.

2010-01-01

Optimal performance of LC-MS/MS platforms is critical to generating high quality proteomics data. Although individual laboratories have developed quality control samples, there is no widely available performance standard of biological complexity (and associated reference data sets) for benchmarking of platform performance for analysis of complex biological proteomes across different laboratories in the community. Individual preparations of the yeast Saccharomyces cerevisiae proteome have been used extensively by laboratories in the proteomics community to characterize LC-MS platform performance. The yeast proteome is uniquely attractive as a performance standard because it is the most extensively characterized complex biological proteome and the only one associated with several large scale studies estimating the abundance of all detectable proteins. In this study, we describe a standard operating protocol for large scale production of the yeast performance standard and offer aliquots to the community through the National Institute of Standards and Technology where the yeast proteome is under development as a certified reference material to meet the long term needs of the community. Using a series of metrics that characterize LC-MS performance, we provide a reference data set demonstrating typical performance of commonly used ion trap instrument platforms in expert laboratories; the results provide a basis for laboratories to benchmark their own performance, to improve upon current methods, and to evaluate new technologies. Additionally, we demonstrate how the yeast reference, spiked with human proteins, can be used to benchmark the power of proteomics platforms for detection of differentially expressed proteins at different levels of concentration in a complex matrix, thereby providing a metric to evaluate and minimize preanalytical and analytical variation in comparative proteomics experiments. PMID:19858499

Screening of missing proteins in the human liver proteome by improved MRM-approach-based targeted proteomics.

PubMed

Chen, Chen; Liu, Xiaohui; Zheng, Weimin; Zhang, Lei; Yao, Jun; Yang, Pengyuan

2014-04-04

To completely annotate the human genome, the task of identifying and characterizing proteins that currently lack mass spectrometry (MS) evidence is inevitable and urgent. In this study, as the first effort to screen missing proteins in large scale, we developed an approach based on SDS-PAGE followed by liquid chromatography-multiple reaction monitoring (LC-MRM), for screening of those missing proteins with only a single peptide hit in the previous liver proteome data set. Proteins extracted from normal human liver were separated in SDS-PAGE and digested in split gel slice, and the resulting digests were then subjected to LC-schedule MRM analysis. The MRM assays were developed through synthesized crude peptides for target peptides. In total, the expressions of 57 target proteins were confirmed from 185 MRM assays in normal human liver tissues. Among the proved 57 one-hit wonders, 50 proteins are of the minimally redundant set in the PeptideAtlas database, 7 proteins even have none MS-based information previously in various biological processes. We conclude that our SDS-PAGE-MRM workflow can be a powerful approach to screen missing or poorly characterized proteins in different samples and to provide their quantity if detected. The MRM raw data have been uploaded to ISB/SRM Atlas/PASSEL (PXD000648).

Proteomics of blood-based therapeutics: a promising tool for quality assurance in transfusion medicine.

PubMed

Thiele, Thomas; Steil, Leif; Völker, Uwe; Greinacher, Andreas

2007-01-01

Blood-based therapeutics are cellular or plasma components derived from human blood. Their production requires appropriate selection and treatment of the donor and processing of cells or plasma proteins. In contrast to clearly defined, chemically synthesized drugs, blood-derived therapeutics are highly complex mixtures of plasma proteins or even more complex cells. Pathogen transmission by the product as well as changes in the integrity of blood constituents resulting in loss of function or immune modulation are currently important issues in transfusion medicine. Protein modifications can occur during various steps of the production process, such as acquisition, enrichment of separate components (e.g. coagulation factors, cell populations), virus inactivation, conservation, and storage. Contemporary proteomic strategies allow a comprehensive assessment of protein modifications with high coverage, offer capabilities for qualitative and even quantitative analysis, and for high-throughput protein identification. Traditionally, proteomics approaches predominantly relied on two-dimensional gel electrophoresis (2-DE). Even if 2-DE is still state of the art, it has inherent limitations that are mainly based on the physicochemical properties of the proteins analyzed; for example, proteins with extremes in molecular mass and hydrophobicity (most membrane proteins) are difficult to assess by 2-DE. These limitations have fostered the development of mass spectrometry centered on non-gel-based separation approaches, which have proven to be highly successful and are thus complementing and even partially replacing 2-DE-based approaches. Although blood constituents have been extensively analyzed by proteomics, this technology has not been widely applied to assess or even improve blood-derived therapeutics, or to monitor the production processes. As proteomic technologies have the capacity to provide comprehensive information about changes occurring during processing and storage of blood products, proteomics can potentially guide improvement of pathogen inactivation procedures and engineering of stem cells, and may also allow a better understanding of factors influencing the immunogenicity of blood-derived therapeutics. An important development in proteomics is the reduction of inter-assay variability. This now allows the screening of samples taken from the same product over time or before and after processing. Optimized preparation procedures and storage conditions will reduce the risk of protein alterations, which in turn may contribute to better recovery, reduced exposure to allogeneic proteins, and increased transfusion safety.

A draft map of the mouse pluripotent stem cell spatial proteome

PubMed Central

Christoforou, Andy; Mulvey, Claire M.; Breckels, Lisa M.; Geladaki, Aikaterini; Hurrell, Tracey; Hayward, Penelope C.; Naake, Thomas; Gatto, Laurent; Viner, Rosa; Arias, Alfonso Martinez; Lilley, Kathryn S.

2016-01-01

Knowledge of the subcellular distribution of proteins is vital for understanding cellular mechanisms. Capturing the subcellular proteome in a single experiment has proven challenging, with studies focusing on specific compartments or assigning proteins to subcellular niches with low resolution and/or accuracy. Here we introduce hyperLOPIT, a method that couples extensive fractionation, quantitative high-resolution accurate mass spectrometry with multivariate data analysis. We apply hyperLOPIT to a pluripotent stem cell population whose subcellular proteome has not been extensively studied. We provide localization data on over 5,000 proteins with unprecedented spatial resolution to reveal the organization of organelles, sub-organellar compartments, protein complexes, functional networks and steady-state dynamics of proteins and unexpected subcellular locations. The method paves the way for characterizing the impact of post-transcriptional and post-translational modification on protein location and studies involving proteome-level locational changes on cellular perturbation. An interactive open-source resource is presented that enables exploration of these data. PMID:26754106

TrSDB: a proteome database of transcription factors

PubMed Central

Hermoso, Antoni; Aguilar, Daniel; Aviles, Francesc X.; Querol, Enrique

2004-01-01

TrSDB—TranScout Database—(http://ibb.uab.es/trsdb) is a proteome database of eukaryotic transcription factors based upon predicted motifs by TranScout and data sources such as InterPro and Gene Ontology Annotation. Nine eukaryotic proteomes are included in the current version. Extensive and diverse information for each database entry, different analyses considering TranScout classification and similarity relationships are offered for research on transcription factors or gene expression. PMID:14681387

Clinical potential of proteomics in the diagnosis of ovarian cancer.

PubMed

Ardekani, Ali M; Liotta, Lance A; Petricoin, Emanuel F

2002-07-01

The need for specific and sensitive markers of ovarian cancer is critical. Finding a sensitive and specific test for its detection has an important public health impact. Currently, there are no effective screening options available for patients with ovarian cancer. CA-125, the most widely used biomarker for ovarian cancer, does not have a high positive predictive value and it is only effective when used in combination with other diagnostic tests. However, pathologic changes taking place within the ovary may be reflected in biomarker patterns in the serum. Combination of mass spectra generated by new proteomic technologies, such as surface-enhanced laser desorption ionization time-of-flight (SELDI-TOF) and artificial-intelligence-based informatic algorithms, have been used to discover a small set of key protein values and discriminate normal from ovarian cancer patients. Serum proteomic pattern analysis might be applied ultimately in medical screening clinics, as a supplement to the diagnostic work-up and evaluation.

QPROT: Statistical method for testing differential expression using protein-level intensity data in label-free quantitative proteomics.

PubMed

Choi, Hyungwon; Kim, Sinae; Fermin, Damian; Tsou, Chih-Chiang; Nesvizhskii, Alexey I

2015-11-03

We introduce QPROT, a statistical framework and computational tool for differential protein expression analysis using protein intensity data. QPROT is an extension of the QSPEC suite, originally developed for spectral count data, adapted for the analysis using continuously measured protein-level intensity data. QPROT offers a new intensity normalization procedure and model-based differential expression analysis, both of which account for missing data. Determination of differential expression of each protein is based on the standardized Z-statistic based on the posterior distribution of the log fold change parameter, guided by the false discovery rate estimated by a well-known Empirical Bayes method. We evaluated the classification performance of QPROT using the quantification calibration data from the clinical proteomic technology assessment for cancer (CPTAC) study and a recently published Escherichia coli benchmark dataset, with evaluation of FDR accuracy in the latter. QPROT is a statistical framework with computational software tool for comparative quantitative proteomics analysis. It features various extensions of QSPEC method originally built for spectral count data analysis, including probabilistic treatment of missing values in protein intensity data. With the increasing popularity of label-free quantitative proteomics data, the proposed method and accompanying software suite will be immediately useful for many proteomics laboratories. This article is part of a Special Issue entitled: Computational Proteomics. Copyright © 2015 Elsevier B.V. All rights reserved.

CrossCheck: an open-source web tool for high-throughput screen data analysis.

PubMed

Najafov, Jamil; Najafov, Ayaz

2017-07-19

Modern high-throughput screening methods allow researchers to generate large datasets that potentially contain important biological information. However, oftentimes, picking relevant hits from such screens and generating testable hypotheses requires training in bioinformatics and the skills to efficiently perform database mining. There are currently no tools available to general public that allow users to cross-reference their screen datasets with published screen datasets. To this end, we developed CrossCheck, an online platform for high-throughput screen data analysis. CrossCheck is a centralized database that allows effortless comparison of the user-entered list of gene symbols with 16,231 published datasets. These datasets include published data from genome-wide RNAi and CRISPR screens, interactome proteomics and phosphoproteomics screens, cancer mutation databases, low-throughput studies of major cell signaling mediators, such as kinases, E3 ubiquitin ligases and phosphatases, and gene ontological information. Moreover, CrossCheck includes a novel database of predicted protein kinase substrates, which was developed using proteome-wide consensus motif searches. CrossCheck dramatically simplifies high-throughput screen data analysis and enables researchers to dig deep into the published literature and streamline data-driven hypothesis generation. CrossCheck is freely accessible as a web-based application at http://proteinguru.com/crosscheck.

The Escherichia coli Peripheral Inner Membrane Proteome*

PubMed Central

Papanastasiou, Malvina; Orfanoudaki, Georgia; Koukaki, Marina; Kountourakis, Nikos; Sardis, Marios Frantzeskos; Aivaliotis, Michalis; Karamanou, Spyridoula; Economou, Anastassios

2013-01-01

Biological membranes are essential for cell viability. Their functional characteristics strongly depend on their protein content, which consists of transmembrane (integral) and peripherally associated membrane proteins. Both integral and peripheral inner membrane proteins mediate a plethora of biological processes. Whereas transmembrane proteins have characteristic hydrophobic stretches and can be predicted using bioinformatics approaches, peripheral inner membrane proteins are hydrophilic, exist in equilibria with soluble pools, and carry no discernible membrane targeting signals. We experimentally determined the cytoplasmic peripheral inner membrane proteome of the model organism Escherichia coli using a multidisciplinary approach. Initially, we extensively re-annotated the theoretical proteome regarding subcellular localization using literature searches, manual curation, and multi-combinatorial bioinformatics searches of the available databases. Next we used sequential biochemical fractionations coupled to direct identification of individual proteins and protein complexes using high resolution mass spectrometry. We determined that the proposed cytoplasmic peripheral inner membrane proteome occupies a previously unsuspected ∼19% of the basic E. coli BL21(DE3) proteome, and the detected peripheral inner membrane proteome occupies ∼25% of the estimated expressed proteome of this cell grown in LB medium to mid-log phase. This value might increase when fleeting interactions, not studied here, are taken into account. Several proteins previously regarded as exclusively cytoplasmic bind membranes avidly. Many of these proteins are organized in functional or/and structural oligomeric complexes that bind to the membrane with multiple interactions. Identified proteins cover the full spectrum of biological activities, and more than half of them are essential. Our data suggest that the cytoplasmic proteome displays remarkably dynamic and extensive communication with biological membrane surfaces that we are only beginning to decipher. PMID:23230279

The Escherichia coli Proteome: Past, Present, and Future Prospects†

PubMed Central

Han, Mee-Jung; Lee, Sang Yup

2006-01-01

Proteomics has emerged as an indispensable methodology for large-scale protein analysis in functional genomics. The Escherichia coli proteome has been extensively studied and is well defined in terms of biochemical, biological, and biotechnological data. Even before the entire E. coli proteome was fully elucidated, the largest available data set had been integrated to decipher regulatory circuits and metabolic pathways, providing valuable insights into global cellular physiology and the development of metabolic and cellular engineering strategies. With the recent advent of advanced proteomic technologies, the E. coli proteome has been used for the validation of new technologies and methodologies such as sample prefractionation, protein enrichment, two-dimensional gel electrophoresis, protein detection, mass spectrometry (MS), combinatorial assays with n-dimensional chromatographies and MS, and image analysis software. These important technologies will not only provide a great amount of additional information on the E. coli proteome but also synergistically contribute to other proteomic studies. Here, we review the past development and current status of E. coli proteome research in terms of its biological, biotechnological, and methodological significance and suggest future prospects. PMID:16760308

Dissecting plasmodesmata molecular composition by mass spectrometry-based proteomics.

PubMed

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.

CPTC and NIST-sponsored Yeast Reference Material Now Publicly Available | Office of Cancer Clinical Proteomics Research

Cancer.gov

The yeast protein extract (RM8323) developed by National Institute of Standards and Technology (NIST) under the auspices of NCI's CPTC initiative is currently available to the public at https://www-s.nist.gov/srmors/view_detail.cfm?srm=8323. The yeast proteome offers researchers a unique biological reference material. RM8323 is the most extensively characterized complex biological proteome and the only one associated with several large-scale studies to estimate protein abundance across a wide concentration range.

Scientific Workflow Management in Proteomics

PubMed Central

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 meets blue biotechnology: a wealth of novelties and opportunities.

PubMed

Hartmann, Erica M; Durighello, Emie; Pible, Olivier; Nogales, Balbina; Beltrametti, Fabrizio; Bosch, Rafael; Christie-Oleza, Joseph A; Armengaud, Jean

2014-10-01

Blue biotechnology, in which aquatic environments provide the inspiration for various products such as food additives, aquaculture, biosensors, green chemistry, bioenergy, and pharmaceuticals, holds enormous promise. Large-scale efforts to sequence aquatic genomes and metagenomes, as well as campaigns to isolate new organisms and culture-based screenings, are helping to push the boundaries of known organisms. Mass spectrometry-based proteomics can complement 16S gene sequencing in the effort to discover new organisms of potential relevance to blue biotechnology by facilitating the rapid screening of microbial isolates and by providing in depth profiles of the proteomes and metaproteomes of marine organisms, both model cultivable isolates and, more recently, exotic non-cultivable species and communities. Proteomics has already contributed to blue biotechnology by identifying aquatic proteins with potential applications to food fermentation, the textile industry, and biomedical drug development. In this review, we discuss historical developments in blue biotechnology, the current limitations to the known marine biosphere, and the ways in which mass spectrometry can expand that knowledge. We further speculate about directions that research in blue biotechnology will take given current and near-future technological advancements in mass spectrometry. Copyright © 2014 Elsevier B.V. All rights reserved.

A PROTEOMIC (SELDI-TOF-MS) APPROACH TO ESTROGEN AGONIST SCREENING

EPA Science Inventory

A small fish model and surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI) were used to investigate plasma protein expression as a means to screen chemicals for estrogenic activity. Adult male sheepshead minnows (Cyprinodon variegatus) were place...

A proteomic analysis of the chromoplasts isolated from sweet orange fruits [Citrus sinensis (L.) Osbeck].

PubMed

Zeng, Yunliu; Pan, Zhiyong; Ding, Yuduan; Zhu, Andan; Cao, Hongbo; Xu, Qiang; Deng, Xiuxin

2011-11-01

Here, a comprehensive proteomic analysis of the chromoplasts purified from sweet orange using Nycodenz density gradient centrifugation is reported. A GeLC-MS/MS shotgun approach was used to identify the proteins of pooled chromoplast samples. A total of 493 proteins were identified from purified chromoplasts, of which 418 are putative plastid proteins based on in silico sequence homology and functional analyses. Based on the predicted functions of these identified plastid proteins, a large proportion (∼60%) of the chromoplast proteome of sweet orange is constituted by proteins involved in carbohydrate metabolism, amino acid/protein synthesis, and secondary metabolism. Of note, HDS (hydroxymethylbutenyl 4-diphosphate synthase), PAP (plastid-lipid-associated protein), and psHSPs (plastid small heat shock proteins) involved in the synthesis or storage of carotenoid and stress response are among the most abundant proteins identified. A comparison of chromoplast proteomes between sweet orange and tomato suggested a high level of conservation in a broad range of metabolic pathways. However, the citrus chromoplast was characterized by more extensive carotenoid synthesis, extensive amino acid synthesis without nitrogen assimilation, and evidence for lipid metabolism concerning jasmonic acid synthesis. In conclusion, this study provides an insight into the major metabolic pathways as well as some unique characteristics of the sweet orange chromoplasts at the whole proteome level.

PROTEOMICS IN ECOTOXICOLOGY: PROTEIN EXPRESSION PROFILING TO SCREEN CHEMICALS FOR ENDOCRINE ACTIVITY

EPA Science Inventory

Abstract for poster.

Current endocrine testing methods are animal intensive and lack the throughput necessary to screen large numbers of environmental chemicals for adverse effects. In this study, Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry...

An Extended Proteome Map of the Lysosomal Membrane Reveals Novel Potential Transporters*

PubMed Central

Chapel, Agnès; Kieffer-Jaquinod, Sylvie; Sagné, Corinne; Verdon, Quentin; Ivaldi, Corinne; Mellal, Mourad; Thirion, Jaqueline; Jadot, Michel; Bruley, Christophe; Garin, Jérôme; Gasnier, Bruno; Journet, Agnès

2013-01-01

Lysosomes are membrane-bound endocytic organelles that play a major role in degrading cell macromolecules and recycling their building blocks. A comprehensive knowledge of the lysosome function requires an extensive description of its content, an issue partially addressed by previous proteomic analyses. However, the proteins underlying many lysosomal membrane functions, including numerous membrane transporters, remain unidentified. We performed a comparative, semi-quantitative proteomic analysis of rat liver lysosome-enriched and lysosome-nonenriched membranes and used spectral counts to evaluate the relative abundance of proteins. Among a total of 2,385 identified proteins, 734 proteins were significantly enriched in the lysosomal fraction, including 207 proteins already known or predicted as endo-lysosomal and 94 proteins without any known or predicted subcellular localization. The remaining 433 proteins had been previously assigned to other subcellular compartments but may in fact reside on lysosomes either predominantly or as a secondary location. Many membrane-associated complexes implicated in diverse processes such as degradation, membrane trafficking, lysosome biogenesis, lysosome acidification, signaling, and nutrient sensing were enriched in the lysosomal fraction. They were identified to an unprecedented extent as most, if not all, of their subunits were found and retained by our screen. Numerous transporters were also identified, including 46 novel potentially lysosomal proteins. We expressed 12 candidates in HeLa cells and observed that most of them colocalized with the lysosomal marker LAMP1, thus confirming their lysosomal residency. This list of candidate lysosomal proteins substantially increases our knowledge of the lysosomal membrane and provides a basis for further characterization of lysosomal functions. PMID:23436907

Dr. Janie Merkel is interviewed by Ryan Blum and Janice Friend.

PubMed

Merkel, Janie

2007-12-01

Dr. Janie Merkel is the director of Yale's Chemical Genomics Screening Facility, a high-throughput screening laboratory that is part of the Yale University Center for Genomics and Proteomics. The Screening Facility connects Yale researchers with industry-quality robotic machinery and a diverse group of compound libraries, which have been used successfully to link therapeutic targets with potential therapies.

Analysis of cellular responses of macrophages to zinc ions and zinc oxide nanoparticles: a combined targeted and proteomic approach.

PubMed

Triboulet, Sarah; Aude-Garcia, Catherine; Armand, Lucie; Gerdil, Adèle; Diemer, Hélène; Proamer, Fabienne; Collin-Faure, Véronique; Habert, Aurélie; Strub, Jean-Marc; Hanau, Daniel; Herlin, Nathalie; Carrière, Marie; Van Dorsselaer, Alain; Rabilloud, Thierry

2014-06-07

Two different zinc oxide nanoparticles, as well as zinc ions, are used to study the cellular responses of the RAW 264 macrophage cell line. A proteomic screen is used to provide a wide view of the molecular effects of zinc, and the most prominent results are cross-validated by targeted studies. Furthermore, the alteration of important macrophage functions (e.g. phagocytosis) by zinc is also investigated. The intracellular dissolution/uptake of zinc is also studied to further characterize zinc toxicity. Zinc oxide nanoparticles dissolve readily in the cells, leading to high intracellular zinc concentrations, mostly as protein-bound zinc. The proteomic screen reveals a rather weak response in the oxidative stress response pathway, but a strong response both in the central metabolism and in the proteasomal protein degradation pathway. Targeted experiments confirm that carbohydrate catabolism and proteasome are critical determinants of sensitivity to zinc, which also induces DNA damage. Conversely, glutathione levels and phagocytosis appear unaffected at moderately toxic zinc concentrations.

Automation, parallelism, and robotics for proteomics.

PubMed

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.

The role of targeted chemical proteomics in pharmacology

PubMed Central

Sutton, Chris W

2012-01-01

Traditionally, proteomics is the high-throughput characterization of the global complement of proteins in a biological system using cutting-edge technologies (robotics and mass spectrometry) and bioinformatics tools (Internet-based search engines and databases). As the field of proteomics has matured, a diverse range of strategies have evolved to answer specific problems. Chemical proteomics is one such direction that provides the means to enrich and detect less abundant proteins (the ‘hidden’ proteome) from complex mixtures of wide dynamic range (the ‘deep’ proteome). In pharmacology, chemical proteomics has been utilized to determine the specificity of drugs and their analogues, for anticipated known targets, only to discover other proteins that bind and could account for side effects observed in preclinical and clinical trials. As a consequence, chemical proteomics provides a valuable accessory in refinement of second- and third-generation drug design for treatment of many diseases. However, determining definitive affinity capture of proteins by a drug immobilized on soft gel chromatography matrices has highlighted some of the challenges that remain to be addressed. Examples of the different strategies that have emerged using well-established drugs against pharmaceutically important enzymes, such as protein kinases, metalloproteases, PDEs, cytochrome P450s, etc., indicate the potential opportunity to employ chemical proteomics as an early-stage screening approach in the identification of new targets. PMID:22074351

Expanding the bovine milk proteome through extensive fractionation.

PubMed

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 different fractions reduced the number to 376 unique proteins in 2 replicates. In addition, 366 proteins were detected by this process in 1 replicate. Hence, by applying different fractionation techniques to milk, we expanded the milk proteome. The milk proteome map may serve as a reference for scientists working in the dairy sector. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

The Multinational Arabidopsis Steering Subcommittee for Proteomics Assembles the Largest Proteome Database Resource for Plant Systems Biology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weckwerth, Wolfram; Baginsky, Sacha; Van Wijk, Klass

2009-12-01

In the past 10 years, we have witnessed remarkable advances in the field of plant molecular biology. The rapid development of proteomic technologies and the speed with which these techniques have been applied to the field have altered our perception of how we can analyze proteins in complex systems. At nearly the same time, the availability of the complete genome for the model plant Arabidopsis thaliana was released; this effort provides an unsurpassed resource for the identification of proteins when researchers use MS to analyze plant samples. Recognizing the growth in this area, the Multinational Arabidopsis Steering Committee (MASC) establishedmore » a subcommittee for A. thaliana proteomics in 2006 with the objective of consolidating databases, technique standards, and experimentally validated candidate genes and functions. Since the establishment of the Multinational Arabidopsis Steering Subcommittee for Proteomics (MASCP), many new approaches and resources have become available. Recently, the subcommittee established a webpage to consolidate this information (www.masc-proteomics.org). It includes links to plant proteomic databases, general information about proteomic techniques, meeting information, a summary of proteomic standards, and other relevant resources. Altogether, this website provides a useful resource for the Arabidopsis proteomics community. In the future, the website will host discussions and investigate the cross-linking of databases. The subcommittee members have extensive experience in arabidopsis proteomics and collectively have produced some of the most extensive proteomics data sets for this model plant (Table S1 in the Supporting Information has a list of resources). The largest collection of proteomics data from a single study in A. thaliana was assembled into an accessible database (AtProteome; http://fgcz-atproteome.unizh.ch/index.php) and was recently published by the Baginsky lab.1 The database provides links to major Arabidopsis online resources, and raw data have been deposited in PRIDE and PRIDE BioMart. Included in this database is an Arabidopsis proteome map that provides evidence for the expression of {approx}50% of all predicted gene models, including several alternative gene models that are not represented in The Arabidopsis Information Resource (TAIR) protein database. A set of organ-specific biomarkers is provided, as well as organ-specific proteotypic peptides for 4105 proteins that can be used to facilitate targeted quantitative proteomic surveys. In the future, the AtProteome database will be linked to additional existing resources developed by MASCP members, such as PPDB, ProMEX, and SUBA. The most comprehensive study on the Arabidopsis chloroplast proteome, which includes information on chloroplast sorting signals, posttranslational modifications (PTMs), and protein abundances (analyzed by high-accuracy MS [Orbitrap]), was recently published by the van Wijk lab.2 These and previous data are available via the plant proteome database (PPDB; http://ppdb.tc.cornell.edu) for A. thaliana and maize. PPDB provides genome-wide experimental and functional characterization of the A. thaliana and maize proteomes, including PTMs and subcellular localization information, with an emphasis on leaf and plastid proteins. Maize and Arabidopsis proteome entries are directly linked via internal BLAST alignments within PPDB. Direct links for each protein to TAIR, SUBA, ProMEX, and other resources are also provided.« less

Proteomics of exhaled breath: methodological nuances and pitfalls.

PubMed

Kurova, Viktoria S; Anaev, Eldar C; Kononikhin, Alexey S; Fedorchenko, Kristina Yu; Popov, Igor A; Kalupov, Timothey L; Bratanov, Dmitriy O; Nikolaev, Eugenie N; Varfolomeev, Sergey D

2009-01-01

The analysis of exhaled breath condensate (EBC) can be an alternative to traditional endoscopic sampling of lower respiratory tract secretions. This is a simple non-invasive method of diagnosing respiratory diseases, in particular, respiratory inflammatory processes. Samples were collected with a special device-condenser (ECoScreen, VIASYS Healthcare, Germany), then treated with trypsin according to the proteomics protocol for standard protein mixtures and analyzed by nanoflow high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) with a 7-Tesla Finnigan LTQ-FT mass spectrometer (Thermo Electron, Germany). Mascot software (Matrixscience) was used for screening the database NCBInr for proteins corresponding to the peptide maps that were obtained. EBCs from 17 young healthy non-smoking donors were collected. Different methods for concentrating protein were compared in order to optimize EBC preparations for proteomic analysis. The procedure that was chosen allowed identification of proteins exhaled by healthy people. The major proteins in the condensates were cytoskeletal keratins. Another 12 proteins were identified in EBC from healthy non-smokers. Some keratins were found in the ambient air and may be considered exogenous components of exhaled air. Knowledge of the normal proteome of exhaled breath allows one to look for biomarkers of different disease states in EBC. Proteins in ambient air can be identified in the respiratory tract and should be excluded from the analysis of the proteome of EBC. The results obtained allowed us to choose the most effective procedure of sample preparation when working with samples containing very low protein concentrations.

GENOMIC AND PROTEOMIC BASIS FOR INTERSPECIES EXTRAPOLATIONS BASED UPON ESTROGEN AND ANDROGEN RECEPTORSTRUCTURE AND FUNCTION AMONG ANIMALS

EPA Science Inventory

Most in vitro hazard assessments for the screening and identification of endocrine disrupting compounds (EDCs), including those outlined in the EDSP Tier 1 Screening (T1S) protocols, use mammalian steroid hormone receptors. There is uncertainty, however, concerning differences th...

Salivary Biomarkers in Cancer Detection

PubMed Central

Wang, Xiaoqian; Kaczor-Urbanowicz, Karolina Elżbieta; Wong, David T.W.

2017-01-01

Cancer is the second most common cause of death in the United States. Its symptoms are often not specific and absent, until the tumors have already metastasized. Therefore, there is an urgent demand for developing rapid, highly accurate and non-invasive tools for cancer screening, early detection, diagnostics, staging and prognostics. Saliva as a multi-constituent oral fluid, comprises secretions from the major and minor salivary glands, extensively supplied by blood. Molecules such as DNAs, RNAs, proteins, metabolites, and microbiota, present in blood, could be also found in saliva. Recently, salivary diagnostics has drawn significant attention for the detection of specific biomarkers, since the sample collection and processing are simple, cost-effective, precise and do not cause patient discomfort. Here, we review recent salivary candidate biomarkers for systemic cancers by dividing them according to their origin into: genomic, transcriptomic, proteomic, metabolomic and microbial types. PMID:27943101

A proteomic analysis of the chromoplasts isolated from sweet orange fruits [Citrus sinensis (L.) Osbeck

PubMed Central

Zeng, Yunliu; Pan, Zhiyong; Ding, Yuduan; Zhu, Andan; Cao, Hongbo; Xu, Qiang; Deng, Xiuxin

2011-01-01

Here, a comprehensive proteomic analysis of the chromoplasts purified from sweet orange using Nycodenz density gradient centrifugation is reported. A GeLC-MS/MS shotgun approach was used to identify the proteins of pooled chromoplast samples. A total of 493 proteins were identified from purified chromoplasts, of which 418 are putative plastid proteins based on in silico sequence homology and functional analyses. Based on the predicted functions of these identified plastid proteins, a large proportion (∼60%) of the chromoplast proteome of sweet orange is constituted by proteins involved in carbohydrate metabolism, amino acid/protein synthesis, and secondary metabolism. Of note, HDS (hydroxymethylbutenyl 4-diphosphate synthase), PAP (plastid-lipid-associated protein), and psHSPs (plastid small heat shock proteins) involved in the synthesis or storage of carotenoid and stress response are among the most abundant proteins identified. A comparison of chromoplast proteomes between sweet orange and tomato suggested a high level of conservation in a broad range of metabolic pathways. However, the citrus chromoplast was characterized by more extensive carotenoid synthesis, extensive amino acid synthesis without nitrogen assimilation, and evidence for lipid metabolism concerning jasmonic acid synthesis. In conclusion, this study provides an insight into the major metabolic pathways as well as some unique characteristics of the sweet orange chromoplasts at the whole proteome level. PMID:21841170

Proteomic analysis of human aqueous humor using multidimensional protein identification technology

PubMed Central

Richardson, Matthew R.; Price, Marianne O.; Price, Francis W.; Pardo, Jennifer C.; Grandin, Juan C.; You, Jinsam; Wang, Mu

2009-01-01

Aqueous humor (AH) supports avascular tissues in the anterior segment of the eye, maintains intraocular pressure, and potentially influences the pathogenesis of ocular diseases. Nevertheless, the AH proteome is still poorly defined despite several previous efforts, which were hindered by interfering high abundance proteins, inadequate animal models, and limited proteomic technologies. To facilitate future investigations into AH function, the AH proteome was extensively characterized using an advanced proteomic approach. Samples from patients undergoing cataract surgery were pooled and depleted of interfering abundant proteins and thereby divided into two fractions: albumin-bound and albumin-depleted. Multidimensional Protein Identification Technology (MudPIT) was utilized for each fraction; this incorporates strong cation exchange chromatography to reduce sample complexity before reversed-phase liquid chromatography and tandem mass spectrometric analysis. Twelve proteins had multi-peptide, high confidence identifications in the albumin-bound fraction and 50 proteins had multi-peptide, high confidence identifications in the albumin-depleted fraction. Gene ontological analyses were performed to determine which cellular components and functions were enriched. Many proteins were previously identified in the AH and for several their potential role in the AH has been investigated; however, the majority of identified proteins were novel and only speculative roles can be suggested. The AH was abundant in anti-oxidant and immunoregulatory proteins as well as anti-angiogenic proteins, which may be involved in maintaining the avascular tissues. This is the first known report to extensively characterize and describe the human AH proteome and lays the foundation for future work regarding its function in homeostatic and pathologic states. PMID:20019884

MAPA distinguishes genotype-specific variability of highly similar regulatory protein isoforms in potato tuber.

PubMed

Hoehenwarter, Wolfgang; Larhlimi, Abdelhalim; Hummel, Jan; Egelhofer, Volker; Selbig, Joachim; van Dongen, Joost T; Wienkoop, Stefanie; Weckwerth, Wolfram

2011-07-01

Mass Accuracy Precursor Alignment is a fast and flexible method for comparative proteome analysis that allows the comparison of unprecedented numbers of shotgun proteomics analyses on a personal computer in a matter of hours. We compared 183 LC-MS analyses and more than 2 million MS/MS spectra and could define and separate the proteomic phenotypes of field grown tubers of 12 tetraploid cultivars of the crop plant Solanum tuberosum. Protein isoforms of patatin as well as other major gene families such as lipoxygenase and cysteine protease inhibitor that regulate tuber development were found to be the primary source of variability between the cultivars. This suggests that differentially expressed protein isoforms modulate genotype specific tuber development and the plant phenotype. We properly assigned the measured abundance of tryptic peptides to different protein isoforms that share extensive stretches of primary structure and thus inferred their abundance. Peptides unique to different protein isoforms were used to classify the remaining peptides assigned to the entire subset of isoforms based on a common abundance profile using multivariate statistical procedures. We identified nearly 4000 proteins which we used for quantitative functional annotation making this the most extensive study of the tuber proteome to date.

Recent advances in stable isotope labeling based techniques for proteome relative quantification.

PubMed

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

2014-10-24

The large scale relative quantification of all proteins expressed in biological samples under different states is of great importance for discovering proteins with important biological functions, as well as screening disease related biomarkers and drug targets. Therefore, the accurate quantification of proteins at proteome level has become one of the key issues in protein science. Herein, the recent advances in stable isotope labeling based techniques for proteome relative quantification were reviewed, from the aspects of metabolic labeling, chemical labeling and enzyme-catalyzed labeling. Furthermore, the future research direction in this field was prospected. Copyright © 2014 Elsevier B.V. All rights reserved.

The online Tabloid Proteome: an annotated database of protein associations

PubMed Central

Turan, Demet; Tavernier, Jan

2018-01-01

Abstract A complete knowledge of the proteome can only be attained by determining the associations between proteins, along with the nature of these associations (e.g. physical contact in protein–protein interactions, participation in complex formation or different roles in the same pathway). Despite extensive efforts in elucidating direct protein interactions, our knowledge on the complete spectrum of protein associations remains limited. We therefore developed a new approach that detects protein associations from identifications obtained after re-processing of large-scale, public mass spectrometry-based proteomics data. Our approach infers protein association based on the co-occurrence of proteins across many different proteomics experiments, and provides information that is almost completely complementary to traditional direct protein interaction studies. We here present a web interface to query and explore the associations derived from this method, called the online Tabloid Proteome. The online Tabloid Proteome also integrates biological knowledge from several existing resources to annotate our derived protein associations. The online Tabloid Proteome is freely available through a user-friendly web interface, which provides intuitive navigation and data exploration options for the user at http://iomics.ugent.be/tabloidproteome. PMID:29040688

LC–MS Proteomics Analysis of the Insulin/IGF-1-Deficient Caenorhabditis elegans daf-2(e1370) Mutant Reveals Extensive Restructuring of Intermediary Metabolism

PubMed Central

2015-01-01

The insulin/IGF-1 receptor is a major known determinant of dauer formation, stress resistance, longevity, and metabolism in Caenorhabditis elegans. In the past, whole-genome transcript profiling was used extensively to study differential gene expression in response to reduced insulin/IGF-1 signaling, including the expression levels of metabolism-associated genes. Taking advantage of the recent developments in quantitative liquid chromatography mass spectrometry (LC–MS)-based proteomics, we profiled the proteomic changes that occur in response to activation of the DAF-16 transcription factor in the germline-less glp-4(bn2);daf-2(e1370) receptor mutant. Strikingly, the daf-2 profile suggests extensive reorganization of intermediary metabolism, characterized by the upregulation of many core intermediary metabolic pathways. These include glycolysis/gluconeogenesis, glycogenesis, pentose phosphate cycle, citric acid cycle, glyoxylate shunt, fatty acid β-oxidation, one-carbon metabolism, propionate and tyrosine catabolism, and complexes I, II, III, and V of the electron transport chain. Interestingly, we found simultaneous activation of reciprocally regulated metabolic pathways, which is indicative of spatiotemporal coordination of energy metabolism and/or extensive post-translational regulation of these enzymes. This restructuring of daf-2 metabolism is reminiscent to that of hypometabolic dauers, allowing the efficient and economical utilization of internal nutrient reserves and possibly also shunting metabolites through alternative energy-generating pathways to sustain longevity. PMID:24555535

Gene and protein expression following exposure to radiofrequency fields from mobile phones.

PubMed

Vanderstraeten, Jacques; Verschaeve, Luc

2008-09-01

Since 1999, several articles have been published on genome-wide and/or proteome-wide response after exposure to radiofrequency (RF) fields whose signal and intensities were similar to or typical of those of currently used mobile telephones. These studies were performed using powerful high-throughput screening techniques (HTSTs) of transcriptomics and/or proteomics, which allow for the simultaneous screening of the expression of thousands of genes or proteins. We reviewed these HTST-based studies and compared the results with currently accepted concepts about the effects of RF fields on gene expression. In this article we also discuss these last in light of the recent concept of microwave-assisted chemistry. To date, the results of HTST-based studies of transcriptomics and/or proteomics after exposure to RF fields relevant to human exposure are still inconclusive, as most of the positive reports are flawed by methodologic imperfections or shortcomings. In addition, when positive findings were reported, no precise response pattern could be identified in a reproducible way. In particular, results from HTST studies tend to exclude the role of a cell stressor for exposure to RF fields at nonthermal intensities. However, on the basis of lessons from microwave-assisted chemistry, we can assume that RF fields might affect heat-sensitive gene or protein expression to an extent larger than would be predicted from temperature change only. But in all likelihood, this would concern intensities higher than those relevant to usual human exposure. The precise role of transcriptomics and proteomics in the screening of bioeffects from exposure to RF fields from mobile phones is still uncertain in view of the lack of positively identified phenotypic change and the lack of theoretical, as well as experimental, arguments for specific gene and/or protein response patterns after this kind of exposure.

Drought-Responsive Mechanisms in Plant Leaves Revealed by Proteomics.

PubMed

Wang, Xiaoli; Cai, Xiaofeng; Xu, Chenxi; Wang, Quanhua; Dai, Shaojun

2016-10-18

Plant drought tolerance is a complex trait that requires a global view to understand its underlying mechanism. The proteomic aspects of plant drought response have been extensively investigated in model plants, crops and wood plants. In this review, we summarize recent proteomic studies on drought response in leaves to reveal the common and specialized drought-responsive mechanisms in different plants. Although drought-responsive proteins exhibit various patterns depending on plant species, genotypes and stress intensity, proteomic analyses show that dominant changes occurred in sensing and signal transduction, reactive oxygen species scavenging, osmotic regulation, gene expression, protein synthesis/turnover, cell structure modulation, as well as carbohydrate and energy metabolism. In combination with physiological and molecular results, proteomic studies in leaves have helped to discover some potential proteins and/or metabolic pathways for drought tolerance. These findings provide new clues for understanding the molecular basis of plant drought tolerance.

Drought-Responsive Mechanisms in Plant Leaves Revealed by Proteomics

PubMed Central

Wang, Xiaoli; Cai, Xiaofeng; Xu, Chenxi; Wang, Quanhua; Dai, Shaojun

2016-01-01

Plant drought tolerance is a complex trait that requires a global view to understand its underlying mechanism. The proteomic aspects of plant drought response have been extensively investigated in model plants, crops and wood plants. In this review, we summarize recent proteomic studies on drought response in leaves to reveal the common and specialized drought-responsive mechanisms in different plants. Although drought-responsive proteins exhibit various patterns depending on plant species, genotypes and stress intensity, proteomic analyses show that dominant changes occurred in sensing and signal transduction, reactive oxygen species scavenging, osmotic regulation, gene expression, protein synthesis/turnover, cell structure modulation, as well as carbohydrate and energy metabolism. In combination with physiological and molecular results, proteomic studies in leaves have helped to discover some potential proteins and/or metabolic pathways for drought tolerance. These findings provide new clues for understanding the molecular basis of plant drought tolerance. PMID:27763546

A Combined Omics Approach to Generate the Surface Atlas of Human Naive CD4+ T Cells during Early T-Cell Receptor Activation*

PubMed Central

Graessel, Anke; Hauck, Stefanie M.; von Toerne, Christine; Kloppmann, Edda; Goldberg, Tatyana; Koppensteiner, Herwig; Schindler, Michael; Knapp, Bettina; Krause, Linda; Dietz, Katharina; Schmidt-Weber, Carsten B.; Suttner, Kathrin

2015-01-01

Naive CD4+ T cells are the common precursors of multiple effector and memory T-cell subsets and possess a high plasticity in terms of differentiation potential. This stem-cell-like character is important for cell therapies aiming at regeneration of specific immunity. Cell surface proteins are crucial for recognition and response to signals mediated by other cells or environmental changes. Knowledge of cell surface proteins of human naive CD4+ T cells and their changes during the early phase of T-cell activation is urgently needed for a guided differentiation of naive T cells and may support the selection of pluripotent cells for cell therapy. Periodate oxidation and aniline-catalyzed oxime ligation technology was applied with subsequent quantitative liquid chromatography-tandem MS to generate a data set describing the surface proteome of primary human naive CD4+ T cells and to monitor dynamic changes during the early phase of activation. This led to the identification of 173 N-glycosylated surface proteins. To independently confirm the proteomic data set and to analyze the cell surface by an alternative technique a systematic phenotypic expression analysis of surface antigens via flow cytometry was performed. This screening expanded the previous data set, resulting in 229 surface proteins, which were expressed on naive unstimulated and activated CD4+ T cells. Furthermore, we generated a surface expression atlas based on transcriptome data, experimental annotation, and predicted subcellular localization, and correlated the proteomics result with this transcriptional data set. This extensive surface atlas provides an overall naive CD4+ T cell surface resource and will enable future studies aiming at a deeper understanding of mechanisms of T-cell biology allowing the identification of novel immune targets usable for the development of therapeutic treatments. PMID:25991687

Functional protease profiling for diagnosis of malignant disease.

PubMed

Findeisen, Peter; Neumaier, Michael

2012-01-01

Clinical proteomic profiling by mass spectrometry (MS) aims at uncovering specific alterations within mass profiles of clinical specimens that are of diagnostic value for the detection and classification of various diseases including cancer. However, despite substantial progress in the field, the clinical proteomic profiling approaches have not matured into routine diagnostic applications so far. Their limitations are mainly related to high-abundance proteins and their complex processing by a multitude of endogenous proteases thus making rigorous standardization difficult. MS is biased towards the detection of low-molecular-weight peptides. Specifically, in serum specimens, the particular fragments of proteolytically degraded proteins are amenable to MS analysis. Proteases are known to be involved in tumour progression and tumour-specific proteases are released into the blood stream presumably as a result of invasive progression and metastasis. Thus, the determination of protease activity in clinical specimens from patients with malignant disease can offer diagnostic and also therapeutic options. The identification of specific substrates for tumour proteases in complex biological samples is challenging, but proteomic screens for proteases/substrate interactions are currently experiencing impressive progress. Such proteomic screens include peptide-based libraries, differential isotope labelling in combination with MS, quantitative degradomic analysis of proteolytically generated neo-N-termini, monitoring the degradation of exogenous reporter peptides with MS, and activity-based protein profiling. In the present article, we summarize and discuss the current status of proteomic techniques to identify tumour-specific protease-substrate interactions for functional protease profiling. Thereby, we focus on the potential diagnostic use of the respective approaches. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteomics and syndrome of Chinese medicine

PubMed Central

Lu, Chuan-Li; Qv, Xiao-Ying; Jiang, Jian-Guo

2010-01-01

Abstract Syndrome of Chinese medicine is an understanding of the regularity of disease occurrence and development and its performance of symptoms. Syndrome is the key to recognize diseases and the foundation to treat them. However, because of the complexity of the concept and the limitation of present investigations, the research of syndrome is hard to go further. Proteomics has been received extensive attention in the area of medical diagnosis and drug development. In the holistic and systemic context, proteomics have a convergence with traditional Chinese medicine (TCM) syndrome, which could overcome the one-sidedness and singleness of TCM and avoid the complication and tedious processes. Chinese medicine has a wealth of experience and proteomics has a substantial research potential, the integration of the two aspects will bring a great enhancement of our knowledge of disease. PMID:20874721

Selective staining of proteins with hydrophobic surface sites on a native electrophoretic gel.

PubMed

Bertsch, Martina; Kassner, Richard J

2003-01-01

Chemical proteomics aims to characterize all of the proteins in the proteome with respect to their function, which is associated with their interaction with other molecules. We propose the identification of a subproteomic library of expressed proteins whose native structures are typified by the presence of hydrophobic surface sites, which are often involved in interactions with small molecules, membrane lipids, and other proteins, pertaining to their functions. We demonstrate that soluble globular proteins with hydrophobic surface sites can be detected selectively by staining on an electrophoretic gel run under nondenaturing conditions. The application of these staining techniques may help elucidate new catalytic, transport, and regulatory functionalities in complex proteomic screenings.

Extensible byssus of Pinctada fucata: Ca2+-stabilized nanocavities and a thrombospondin-1 protein

NASA Astrophysics Data System (ADS)

Liu, Chuang; Li, Shiguo; Huang, Jingliang; Liu, Yangjia; Jia, Ganchu; Xie, Liping; Zhang, Rongqing

2015-10-01

The extensible byssus is produced by the foot of bivalve animals, including the pearl oyster Pinctada fucata, and enables them to attach to hard underwater surfaces. However, the mechanism of their extensibility is not well understood. To understand this mechanism, we analyzed the ultrastructure, composition and mechanical properties of the P. fucata byssus using electron microscopy, elemental analysis, proteomics and mechanical testing. In contrast to the microstructures of Mytilus sp. byssus, the P. fucata byssus has an exterior cuticle without granules and an inner core with nanocavities. The removal of Ca2+ by ethylenediaminetetraacetic acid (EDTA) treatment expands the nanocavities and reduces the extensibility of the byssus, which is accompanied by a decrease in the β-sheet conformation of byssal proteins. Through proteomic methods, several proteins with antioxidant and anti-corrosive properties were identified as the main components of the distal byssus regions. Specifically, a protein containing thrombospondin-1 (TSP-1), which is highly expressed in the foot, is hypothesized to be responsible for byssus extensibility. Together, our findings demonstrate the importance of inorganic ions and multiple proteins for bivalve byssus extension, which could guide the future design of biomaterials for use in seawater.

Extensible byssus of Pinctada fucata: Ca2+-stabilized nanocavities and a thrombospondin-1 protein

PubMed Central

Liu, Chuang; Li, Shiguo; Huang, Jingliang; Liu, Yangjia; Jia, Ganchu; Xie, Liping; Zhang, Rongqing

2015-01-01

The extensible byssus is produced by the foot of bivalve animals, including the pearl oyster Pinctada fucata, and enables them to attach to hard underwater surfaces. However, the mechanism of their extensibility is not well understood. To understand this mechanism, we analyzed the ultrastructure, composition and mechanical properties of the P. fucata byssus using electron microscopy, elemental analysis, proteomics and mechanical testing. In contrast to the microstructures of Mytilus sp. byssus, the P. fucata byssus has an exterior cuticle without granules and an inner core with nanocavities. The removal of Ca2+ by ethylenediaminetetraacetic acid (EDTA) treatment expands the nanocavities and reduces the extensibility of the byssus, which is accompanied by a decrease in the β-sheet conformation of byssal proteins. Through proteomic methods, several proteins with antioxidant and anti-corrosive properties were identified as the main components of the distal byssus regions. Specifically, a protein containing thrombospondin-1 (TSP-1), which is highly expressed in the foot, is hypothesized to be responsible for byssus extensibility. Together, our findings demonstrate the importance of inorganic ions and multiple proteins for bivalve byssus extension, which could guide the future design of biomaterials for use in seawater. PMID:26446436

Global Proteomics Analysis of Protein Lysine Methylation.

PubMed

Cao, Xing-Jun; Garcia, Benjamin A

2016-11-01

Lysine methylation is a common protein post-translational modification dynamically mediated by protein lysine methyltransferases (PKMTs) and protein lysine demethylases (PKDMs). Beyond histone proteins, lysine methylation on non-histone proteins plays a substantial role in a variety of functions in cells and is closely associated with diseases such as cancer. A large body of evidence indicates that the dysregulation of some PKMTs leads to tumorigenesis via their non-histone substrates. However, most studies on other PKMTs have made slow progress owing to the lack of approaches for extensive screening of lysine methylation sites. However, recently, there has been a series of publications to perform large-scale analysis of protein lysine methylation. In this unit, we introduce a protocol for the global analysis of protein lysine methylation in cells by means of immunoaffinity enrichment and mass spectrometry. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

The Escherichia coli O157:H7 cattle immuno-proteome includes outer membrane protein A (OmpA), a modulator of adherence to bovine recto-anal junction squamous epithelial (RSE) cells

PubMed Central

Kudva, Indira T.; Krastins, Bryan; Torres, Alfredo G.; Griffin, Robert W.; Sheng, Haiqing; Sarracino, David A.; Hovde, Carolyn J.; Calderwood, Stephen B.; John, Manohar

2015-01-01

SUMMARY Building on previous studies, we defined the repertoire of proteins comprising the immuno-proteome of E. coli O157:H7 (O157) cultured in DMEM supplemented with norepinephrine (NE; O157 immuno-proteome), a β-adrenergic hormone that regulates E. coli O157 gene expression in the gastrointestinal tract, using a variation of a novel proteomics-based platform proteome mining tool for antigen discovery, called Proteomics-based Expression Library Screening (PELS; Kudva et al., 2006). The E. coli O157 immuno-proteome (O157-IP) comprised 91 proteins, and included those identified previously using PELS, and also proteins comprising DMEM- and bovine rumen fluid- proteomes. Outer membrane protein A (OmpA), a common component of the above proteomes, and reportedly a contributor to E. coli O157 adherence to cultured Hep-2 epithelial cells, was interestingly found to be a modulator rather than a contributor to E. coli O157 adherence to bovine recto-anal junction squamous epithelial (RSE) cells. Our results point to a role for yet to be identified members of the O157-IP in E. coli O157 adherence to RSE-cells, and additionally implicate a possible role for the OmpA regulator, TdcA, in the expression of such adhesins. Our observations have implications for development of efficacious vaccines for preventing E. coli O157 colonization of the bovine gastrointestinal tract. PMID:25643951

A New Mass Spectrometry-compatible Degradable Surfactant for Tissue Proteomics

PubMed Central

Chang, Ying-Hua; Gregorich, Zachery R.; Chen, Albert J.; Hwang, Leekyoung; Guner, Huseyin; Yu, Deyang; Zhang, Jianyi; Ge, Ying

2015-01-01

Tissue proteomics is increasingly recognized for its role in biomarker discovery and disease mechanism investigation. However, protein solubility remains a significant challenge in mass spectrometry (MS)-based tissue proteomics. Conventional surfactants such as sodium dodecyl sulfate (SDS), the preferred surfactant for protein solubilization, are not compatible with MS. Herein, we have screened a library of surfactant-like compounds and discovered an MS-compatible degradable surfactant (MaSDeS) for tissue proteomics that solubilizes all categories of proteins with performance comparable to SDS. The use of MaSDeS in the tissue extraction significantly improves the total number of protein identifications from commonly used tissues, including tissue from the heart, liver, and lung. Notably, MaSDeS significantly enriches membrane proteins, which are often under-represented in proteomics studies. The acid degradable nature of MaSDeS makes it amenable for high-throughput mass spectrometry-based proteomics. In addition, the thermostability of MaSDeS allows for its use in experiments requiring high temperature to facilitate protein extraction and solubilization. Furthermore, we have shown that MaSDeS outperforms the other MS-compatible surfactants in terms of overall protein solubility and the total number of identified proteins in tissue proteomics. Thus, the use of MaSDeS will greatly advance tissue proteomics and realize its potential in basic biomedical and clinical research. MaSDeS could be utilized in a variety of proteomics studies as well as general biochemical and biological experiments that employ surfactants for protein solubilization. PMID:25589168

Proteomic evaluation of milk from different mammalian species as a substitute for breast milk.

PubMed

D'Auria, Enza; Agostoni, Carlo; Giovannini, Marcello; Riva, Enrica; Zetterström, Rolf; Fortin, Riccardo; Greppi, Gian Franco; Bonizzi, Luigi; Roncada, Paola

2005-12-01

As milk represents the main source of nutrition for infants, the question of an effective human milk substitute becomes mandatory when a formula-fed baby is allergic to cows' milk proteins. In this case, formulas containing extensively hydrolysed milk proteins should be preferred, but even such a formula may cause allergic reactions in highly sensitive patients. If there is evidence of cows' milk allergy with IgE-associated symptoms, after 6 mo of age, a soy bean formula may be recommended only when tolerance to soy protein has been established by clinical challenge. In infants with allergic reactions to cows' milk proteins, even after extensive hydrolyzation, proteomic techniques coupled to immunological methods may make it possible to select other milk products that do not contain the same allergens as ordinary cow's milk. In this paper, evidence will be presented that proteomic evaluation of proteins from different mammalian species may be a suitable method of testing whether proteins from the milk of different mammalian species may be used as a substitute for untreated bovine milk. Proteomic evaluation of milk from different mammalian species may not only be of help when recommending suitable feeding in cases of cows' milk allergy but also gives new insight into the background to allergic reactions caused by milk proteins.

Comparative proteomics of matrix fractions between pimpled and normal chicken eggshells.

PubMed

Liu, Zhangguo; Song, Lingzi; Lu, Lizhi; Zhang, Xianfu; Zhang, Fuming; Wang, Kehua; Linhardt, Robert J

2017-09-07

Eggshell matrix can be dissociated into three matrix fractions: acid-insoluble matrix (M1), water-insoluble matrix (M2) and acid-water facultative-soluble matrix (M3). Matrix fractions from pimpled and normal eggshells were compared using label-free proteomic method to understand the differences among three matrix fractions and the proteins involved with eggshell quality. A total of 738 and 600 proteins were identified in the pimpled and normal calcified eggshells, respectively. Both eggshells showed a combined proteomic inventory of 769 proteins. In the same type of eggshell, a high similarity was present in the proteomes of three matrix fractions. These triply overlapped common proteins formed the predominant contributor to proteomic abundance in the matrix fractions. In each matrix fraction and between both eggshell models, normal and pimpled eggshells, a majority of the proteomes of the fractions were commonly observed. Forty-two common major proteins (iBAQ-derived abundance ≥0.095% of proteomic abundance) were identified throughout the three matrix fractions and these proteins might act as backbone constituents in chicken eggshell matrix. Finally, using 1.75-fold as up-regulated and using 0.57-fold as down-regulated cutoff values, twenty-five differential major proteins were screened and they all negatively influence and none showed any effect on eggshell quality. Overall, we uncovered the characteristics of proteomics of three eggshell matrix fractions and identified candidate proteins influencing eggshell quality. The next research on differential proteins will uncover the potential mechanisms underlying how proteins affect eggshell quality. It was reported that the proteins in an eggshell can be divided into insoluble and soluble proteins. The insoluble proteins are thought to be an inter-mineral matrix and acts as a structural framework, while the soluble proteins are thought as intra-mineral matrix that are embedded within the crystal during calcification. However, the difference between matrix fractions is unknown. Cross-analysis of proteomic data of three matrix fractions from the same type of eggshell, uncovered triply overlapped common proteins formed the predominant contributor to proteomic abundance of any matrix fraction, and we suggested that abundance variance of some common proteins between the three matrix fractions might be an important cause of their solubility differences. Moreover, eggshell is formed in hen's uterus, and uterus tend to be considered as unique organ determining eggshell quality. By cross-analysis on proteomic data of three matrix fractions between two eggshell models, normal and pimpled eggshells, the differential proteins were screened as candidates influencing eggshell quality. And we suggested that the liver and spleen or lymphocytes might be the major organs influencing eggshell quality, because the most promising candidates are almost blood and non-collagenous proteins, and originated from above organs. Copyright © 2017 Elsevier B.V. All rights reserved.

LC-MS Proteomics Analysis of the Insulin/IGF-1 Deficient Caenorhabditis elegans daf-2(e1370) Mutant Reveals Extensive Restructuring of Intermediary Metabolism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Depuydt, Geert G.; Xie, Fang; Petyuk, Vladislav A.

2014-02-20

The insulin/IGF-1 receptor is a major known determinant of dauer formation, stress resistance, longevity and metabolism in C. elegans. In the past, whole-genome transcript profiling was used extensively to study differential gene expression in response to reduced insulin/IGF-1 signaling, including expression levels of metabolism-associated genes. Taking advantage of the recent developments in quantitative liquid chromatography mass-spectrometry (LC-MS) based proteomics, we profiled the proteomic changes that occur in response to activation of the DAF-16 transcription factor in the germline-less glp-4(bn2); daf-2(e1370) receptor mutant. Strikingly, the daf-2 profile suggests extensive reorganization of intermediary metabolism, characterized by the up-regulation of many core intermediarymore » metabolic pathways. These include, glycolysis/gluconeogenesis, glycogenesis, pentose phosphate cycle, citric acid cycle, glyoxylate shunt, fatty acid β-oxidation, one-carbon metabolism, propionate and tyrosine catabolism, and complex I, II, III and V of the electron transport chain. Interestingly, we found simultaneous activation of reciprocally regulated metabolic pathways, which is indicative for spatio-temporal coordination of energy metabolism and/or extensive post-translational regulation of these enzymes. This restructuring of daf-2 metabolism is reminiscent to that of hypometabolic dauers, allowing the efficient and economical utilization of internal nutrient reserves, possibly also shunting metabolites through alternative energy-generating pathways, in order to sustain longevity.« less

LC–MS Proteomics Analysis of the Insulin/IGF-1-Deficient Caenorhabditis elegans daf-2(e1370) Mutant Reveals Extensive Restructuring of Intermediary Metabolism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Depuydt, Geert; Xie, Fang; Petyuk, Vladislav A.

2014-04-04

The insulin/IGF-1 receptor is a major known determinant of dauer formation, stress resistance, longevity, and metabolism in Caenorhabditis elegans. In the past, whole-genome transcript profiling was used extensively to study differential gene expression in response to reduced insulin/IGF-1 signaling, including the expression levels of metabolism-associated genes. Taking advantage of the recent developments in quantitative liquid chromatography mass spectrometry (LC–MS)-based proteomics, we profiled the proteomic changes that occur in response to activation of the DAF-16 transcription factor in the germline-less glp-4(bn2);daf-2(e1370) receptor mutant. Strikingly, the daf-2 profile suggests extensive reorganization of intermediary metabolism, characterized by the upregulation of many core intermediarymore » metabolic pathways. These include glycolysis/gluconeogenesis, glycogenesis, pentose phosphate cycle, citric acid cycle, glyoxylate shunt, fatty acid β-oxidation, one-carbon metabolism, propionate and tyrosine catabolism, and complexes I, II, III, and V of the electron transport chain. Interestingly, we found simultaneous activation of reciprocally regulated metabolic pathways, which is indicative of spatiotemporal coordination of energy metabolism and/or extensive post-translational regulation of these enzymes. Finally, this restructuring of daf-2 metabolism is reminiscent to that of hypometabolic dauers, allowing the efficient and economical utilization of internal nutrient reserves and possibly also shunting metabolites through alternative energy-generating pathways to sustain longevity.« less

Five years later: the current status of the use of proteomics and transcriptomics in EMF research.

PubMed

Leszczynski, Dariusz; de Pomerai, David; Koczan, Dirk; Stoll, Dieter; Franke, Helmut; Albar, Juan Pablo

2012-08-01

The World Health Organization's and Radiation and Nuclear Safety Authority's "Workshop on Application of Proteomics and Transcriptomics in Electromagnetic Fields Research" was held in Helsinki in the October/November 2005. As a consequence of this meeting, Proteomics journal published in 2006 a special issue "Application of Proteomics and Transcriptomics in EMF Research" (Vol. 6 No. 17; Guest Editor: D. Leszczynski). This Proteomics issue presented the status of research, of the effects of electromagnetic fields (EMF) using proteomics and transcriptomics methods, present in 2005. The current overview/opinion article presents the status of research in this area by reviewing all studies that were published by the end of 2010. The review work was a part of the European Cooperation in the Field of Scientific and Technical Research (COST) Action BM0704 that created a structure in which researchers in the field of EMF and health shared knowledge and information. The review was prepared by the members of the COST Action BM0704 task group on the high-throughput screening techniques and electromagnetic fields (TG-HTST-EMF). © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Applicability of a high-throughput shotgun plasma protein screening approach in understanding maternal biological pathways relevant to infant birth weight outcome.

PubMed

Kumarathasan, P; Vincent, R; Das, D; Mohottalage, S; Blais, E; Blank, K; Karthikeyan, S; Vuong, N Q; Arbuckle, T E; Fraser, W D

2014-04-04

There are reports linking maternal nutritional status, smoking and environmental chemical exposures to adverse pregnancy outcomes. However, biological bases for association between some of these factors and birth outcomes are yet to be established. The objective of this preliminary work is to test the capability of a new high-throughput shotgun plasma proteomic screening in identifying maternal changes relevant to pregnancy outcome. A subset of third trimester plasma samples (N=12) associated with normal and low-birth weight infants were fractionated, tryptic-digested and analyzed for global proteomic changes using a MALDI-TOF-TOF-MS methodology. Mass spectral data were mined for candidate biomarkers using bioinformatic and statistical tools. Maternal plasma profiles of cytokines (e.g. IL8, TNF-α), chemokines (e.g. MCP-1) and cardiovascular endpoints (e.g. ET-1, MMP-9) were analyzed by a targeted approach using multiplex protein array and HPLC-Fluorescence methods. Target and global plasma proteomic markers were used to identify protein interaction networks and maternal biological pathways relevant to low infant birth weight. Our results exhibited the potential to discriminate specific maternal physiologies relevant to risk of adverse birth outcomes. This proteomic approach can be valuable in understanding the impacts of maternal factors such as environmental contaminant exposures and nutrition on birth outcomes in future work. We demonstrate here the fitness of mass spectrometry-based shot-gun proteomics for surveillance of biological changes in mothers, and for adverse pathway analysis in combination with target biomarker information. This approach has potential for enabling early detection of mothers at risk for low infant birth weight and preterm birth, and thus early intervention for mitigation and prevention of adverse pregnancy outcomes. This article is part of a Special Issue entitled: Can Proteomics Fill the Gap Between Genomics and Phenotypes? Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Bronchoalveolar lavage fluid of lung cancer patients: mapping the uncharted waters using proteomics technology.

PubMed

Oumeraci, Tonio; Schmidt, Bernd; Wolf, Thomas; Zapatka, Marc; Pich, Andreas; Brors, Benedikt; Eils, Roland; Fleischhacker, Michael; Schlegelberger, Brigitte; von Neuhoff, Nils

2011-04-01

The search for proteome-level markers of non-small cell lung cancer (NSCLC) has been mainly limited to serum or cell line screening approaches up to this point. We would like to demonstrate by this proof-of-principle study investigating bronchoalveolar lavage fluid samples from a cohort of NSCLC and control patients, that this readily available biofluid might be a more suitable source for discovering clinically usable NSCLC biomarkers. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Quantitative Analysis of Global Proteome and Lysine Acetylome Reveal the Differential Impacts of VPA and SAHA on HL60 Cells.

PubMed

Zhu, Xiaoyu; Liu, Xin; Cheng, Zhongyi; Zhu, Jun; Xu, Lei; Wang, Fengsong; Qi, Wulin; Yan, Jiawei; Liu, Ning; Sun, Zimin; Liu, Huilan; Peng, Xiaojun; Hao, Yingchan; Zheng, Nan; Wu, Quan

2016-01-29

Valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) are both HDAC inhibitors (HDACi). Previous studies indicated that both inhibitors show therapeutic effects on acute myeloid leukaemia (AML), while the differential impacts of the two different HDACi on AML treatment still remains elusive. In this study, using 3-plex SILAC based quantitative proteomics technique, anti-acetyllysine antibody based affinity enrichment, high resolution LC-MS/MS and intensive bioinformatic analysis, the quantitative proteome and acetylome in SAHA and VPA treated AML HL60 cells were extensively studied. In total, 5,775 proteins and 1,124 lysine acetylation sites were successfully obtained in response to VAP and SAHA treatment. It is found that VPA and SAHA treatment differently induced proteome and acetylome profiling in AML HL60 cells. This study revealed the differential impacts of VPA and SAHA on proteome/acetylome in AML cells, deepening our understanding of HDAC inhibitor mediated AML therapeutics.

Development of Biomarkers for Screening Hepatocellular Carcinoma Using Global Data Mining and Multiple Reaction Monitoring

PubMed Central

Yu, Su Jong; Jang, Eun Sun; Yu, Jiyoung; Cho, Geunhee; Yoon, Jung-Hwan; Kim, Youngsoo

2013-01-01

Hepatocellular carcinoma (HCC) is one of the most common and aggressive cancers and is associated with a poor survival rate. Clinically, the level of alpha-fetoprotein (AFP) has been used as a biomarker for the diagnosis of HCC. The discovery of useful biomarkers for HCC, focused solely on the proteome, has been difficult; thus, wide-ranging global data mining of genomic and proteomic databases from previous reports would be valuable in screening biomarker candidates. Further, multiple reaction monitoring (MRM), based on triple quadrupole mass spectrometry, has been effective with regard to high-throughput verification, complementing antibody-based verification pipelines. In this study, global data mining was performed using 5 types of HCC data to screen for candidate biomarker proteins: cDNA microarray, copy number variation, somatic mutation, epigenetic, and quantitative proteomics data. Next, we applied MRM to verify HCC candidate biomarkers in individual serum samples from 3 groups: a healthy control group, patients who have been diagnosed with HCC (Before HCC treatment group), and HCC patients who underwent locoregional therapy (After HCC treatment group). After determining the relative quantities of the candidate proteins by MRM, we compared their expression levels between the 3 groups, identifying 4 potential biomarkers: the actin-binding protein anillin (ANLN), filamin-B (FLNB), complementary C4-A (C4A), and AFP. The combination of 2 markers (ANLN, FLNB) improved the discrimination of the before HCC treatment group from the healthy control group compared with AFP. We conclude that the combination of global data mining and MRM verification enhances the screening and verification of potential HCC biomarkers. This efficacious integrative strategy is applicable to the development of markers for cancer and other diseases. PMID:23717429

Development of biomarkers for screening hepatocellular carcinoma using global data mining and multiple reaction monitoring.

PubMed

Kim, Hyunsoo; Kim, Kyunggon; Yu, Su Jong; Jang, Eun Sun; Yu, Jiyoung; Cho, Geunhee; Yoon, Jung-Hwan; Kim, Youngsoo

2013-01-01

Hepatocellular carcinoma (HCC) is one of the most common and aggressive cancers and is associated with a poor survival rate. Clinically, the level of alpha-fetoprotein (AFP) has been used as a biomarker for the diagnosis of HCC. The discovery of useful biomarkers for HCC, focused solely on the proteome, has been difficult; thus, wide-ranging global data mining of genomic and proteomic databases from previous reports would be valuable in screening biomarker candidates. Further, multiple reaction monitoring (MRM), based on triple quadrupole mass spectrometry, has been effective with regard to high-throughput verification, complementing antibody-based verification pipelines. In this study, global data mining was performed using 5 types of HCC data to screen for candidate biomarker proteins: cDNA microarray, copy number variation, somatic mutation, epigenetic, and quantitative proteomics data. Next, we applied MRM to verify HCC candidate biomarkers in individual serum samples from 3 groups: a healthy control group, patients who have been diagnosed with HCC (Before HCC treatment group), and HCC patients who underwent locoregional therapy (After HCC treatment group). After determining the relative quantities of the candidate proteins by MRM, we compared their expression levels between the 3 groups, identifying 4 potential biomarkers: the actin-binding protein anillin (ANLN), filamin-B (FLNB), complementary C4-A (C4A), and AFP. The combination of 2 markers (ANLN, FLNB) improved the discrimination of the before HCC treatment group from the healthy control group compared with AFP. We conclude that the combination of global data mining and MRM verification enhances the screening and verification of potential HCC biomarkers. This efficacious integrative strategy is applicable to the development of markers for cancer and other diseases.

Proteomics of ovarian cancer: functional insights and clinical applications

DOE PAGES

Elzek, Mohamed A.; Rodland, Karin D.

2015-03-04

In the past decade, there has been an increasing interest in applying proteomics to assist in understanding the pathogenesis of ovarian cancer, elucidating the mechanism of drug resistance, and in the development of biomarkers for early detection of ovarian cancer. Although ovarian cancer is a spectrum of different diseases, the strategies for diagnosis and treatment with surgery and adjuvant therapy are similar across ovarian cancer types, increasing the general applicability of discoveries made through proteomics research. While proteomic experiments face many difficulties which slow the pace of clinical applications, recent advances in proteomic technology contribute significantly to the identification ofmore » aberrant proteins and networks which can serve as targets for biomarker development and individualized therapies. This review provides a summary of the literature on proteomics’ contributions to ovarian cancer research and highlights the current issues, future directions, and challenges. In conclusion, we propose that protein-level characterization of primary lesion in ovarian cancer can decipher the mystery of this disease, improve diagnostic tools, and lead to more effective screening programs.« less

One Sample, One Shot - Evaluation of sample preparation protocols for the mass spectrometric proteome analysis of human bile fluid without extensive fractionation.

PubMed

Megger, Dominik A; Padden, Juliet; Rosowski, Kristin; Uszkoreit, Julian; Bracht, Thilo; Eisenacher, Martin; Gerges, Christian; Neuhaus, Horst; Schumacher, Brigitte; Schlaak, Jörg F; Sitek, Barbara

2017-02-10

The proteome analysis of bile fluid represents a promising strategy to identify biomarker candidates for various diseases of the hepatobiliary system. However, to obtain substantive results in biomarker discovery studies large patient cohorts necessarily need to be analyzed. Consequently, this would lead to an unmanageable number of samples to be analyzed if sample preparation protocols with extensive fractionation methods are applied. Hence, the performance of simple workflows allowing for "one sample, one shot" experiments have been evaluated in this study. In detail, sixteen different protocols implying modifications at the stages of desalting, delipidation, deglycosylation and tryptic digestion have been examined. Each method has been individually evaluated regarding various performance criteria and comparative analyses have been conducted to uncover possible complementarities. Here, the best performance in terms of proteome coverage has been assessed for a combination of acetone precipitation with in-gel digestion. Finally, a mapping of all obtained protein identifications with putative biomarkers for hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) revealed several proteins easily detectable in bile fluid. These results can build the basis for future studies with large and well-defined patient cohorts in a more disease-related context. Human bile fluid is a proximal body fluid and supposed to be a potential source of disease markers. However, due to its biochemical composition, the proteome analysis of bile fluid still represents a challenging task and is therefore mostly conducted using extensive fractionation procedures. This in turn leads to a high number of mass spectrometric measurements for one biological sample. Considering the fact that in order to overcome the biological variability a high number of biological samples needs to be analyzed in biomarker discovery studies, this leads to the dilemma of an unmanageable number of necessary MS-based analyses. Hence, easy sample preparation protocols are demanded representing a compromise between proteome coverage and simplicity. In the presented study, such protocols have been evaluated regarding various technical criteria (e.g. identification rates, missed cleavages, chromatographic separation) uncovering the strengths and weaknesses of various methods. Furthermore, a cumulative bile proteome list has been generated that extends the current bile proteome catalog by 248 proteins. Finally, a mapping with putative biomarkers for hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) derived from tissue-based studies, revealed several of these proteins being easily and reproducibly detectable in human bile. Therefore, the presented technical work represents a solid base for future disease-related studies. Copyright © 2016 Elsevier B.V. All rights reserved.

Redox proteomics screening cellular factors associated with oxidative stress in hepatocarcinogenesis.

PubMed

Zhou, Li; Wen, Ji; Huang, Zhao; Nice, Edouard C; Huang, Canhua; Zhang, Haiyuan; Li, Qifu

2017-03-01

Liver cancer is a major global health problem being the sixth most common cancer and the third cause of cancer-related death, with hepatocellular carcinoma (HCC) representing more than 90% of primary liver cancers. Mounting evidence suggests that, compared with their normal counterparts, many types of cancer cell have increased levels of ROS. Therefore, cancer cells need to combat high levels of ROS, especially at early stages of tumor development. Recent studies have revealed that ROS-mediated regulation of redox-sensitive proteins (redox sensors) is involved in the pathogenesis and/or progression of many human diseases, including cancer. Unraveling the altered functions of redox sensors and the underlying mechanisms in hepatocarcinogenesis is critical for the development of novel cancer therapeutics. For this reason, redox proteomics has been developed for the high-throughput screening of redox sensors, which will benefit the development of novel therapeutic strategies for the treatment of HCC. In this review, we will briefly introduce several novel redox proteomics techniques that are currently available to study various oxidative modifications in hepatocarcinogenesis and summarize the most important discoveries in the study of redox processes related to the development and progression of HCC. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Role of Proteomics in the Diagnosis and Treatment of Women's Cancers: Current Trends in Technology and Future Opportunities

PubMed Central

Breuer, Eun-Kyoung Yim; Murph, Mandi M.

2011-01-01

Technological and scientific innovations over the last decade have greatly contributed to improved diagnostics, predictive models, and prognosis among cancers affecting women. In fact, an explosion of information in these areas has almost assured future generations that outcomes in cancer will continue to improve. Herein we discuss the current status of breast, cervical, and ovarian cancers as it relates to screening, disease diagnosis, and treatment options. Among the differences in these cancers, it is striking that breast cancer has multiple predictive tests based upon tumor biomarkers and sophisticated, individualized options for prescription therapeutics while ovarian cancer lacks these tools. In addition, cervical cancer leads the way in innovative, cancer-preventative vaccines and multiple screening options to prevent disease progression. For each of these malignancies, emerging proteomic technologies based upon mass spectrometry, stable isotope labeling with amino acids, high-throughput ELISA, tissue or protein microarray techniques, and click chemistry in the pursuit of activity-based profiling can pioneer the next generation of discovery. We will discuss six of the latest techniques to understand proteomics in cancer and highlight research utilizing these techniques with the goal of improvement in the management of women's cancers. PMID:21886869

Proteomic analyses of host and pathogen responses during bovine mastitis.

PubMed

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.

A unique proteomic profile on surface IgM ligation in unmutated chronic lymphocytic leukemia

PubMed Central

Perrot, Aurore; Pionneau, Cédric; Nadaud, Sophie; Davi, Frédéric; Leblond, Véronique; Jacob, Frédéric; Merle-Béral, Hélène; Herbrecht, Raoul; Béné, Marie-Christine; Gribben, John G.; Vallat, Laurent

2011-01-01

Chronic lymphocytic leukemia (CLL) is characterized by a highly variable clinical course with 2 extreme subsets: indolent, ZAP70− and mutated immunoglobulin heavy chain gene (M-CLL); and aggressive, ZAP70+ and unmutated immunoglobulin heavy chain (UM-CLL). Given the long-term suspicion of antigenic stimulation as a primum movens in the disease, the role of the B-cell receptor has been extensively studied in various experimental settings; albeit scarcely in a comparative dynamic proteomic approach. Here we use a quantitative 2-dimensional fluorescence difference gel electrophoresis technology to compare 48 proteomic profiles of the 2 CLL subsets before and after anti-IgM ligation. Differentially expressed proteins were subsequently identified by mass spectrometry. We show that unstimulated M- and UM-CLL cells display distinct proteomic profiles. Furthermore, anti-IgM stimulation induces a specific proteomic response, more pronounced in the more aggressive CLL. Statistical analyses demonstrate several significant protein variations according to stimulation conditions. Finally, we identify an intermediate form of M-CLL cells, with an indolent profile (ZAP70−) but sharing aggressive proteomic profiles alike UM-CLL cells. Collectively, this first quantitative and dynamic proteome analysis of CLL further dissects the complex molecular pathway after B-cell receptor stimulation and depicts distinct proteomic profiles, which could lead to novel molecular stratification of the disease. PMID:21602524

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

PubMed

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.

Gene and Protein Expression following Exposure to Radiofrequency Fields from Mobile Phones

PubMed Central

Vanderstraeten, Jacques; Verschaeve, Luc

2008-01-01

Background Since 1999, several articles have been published on genome-wide and/or proteome-wide response after exposure to radiofrequency (RF) fields whose signal and intensities were similar to or typical of those of currently used mobile telephones. These studies were performed using powerful high-throughput screening techniques (HTSTs) of transcriptomics and/or proteomics, which allow for the simultaneous screening of the expression of thousands of genes or proteins. Objectives We reviewed these HTST-based studies and compared the results with currently accepted concepts about the effects of RF fields on gene expression. In this article we also discuss these last in light of the recent concept of microwave-assisted chemistry. Discussion To date, the results of HTST-based studies of transcriptomics and/or proteomics after exposure to RF fields relevant to human exposure are still inconclusive, as most of the positive reports are flawed by methodologic imperfections or shortcomings. In addition, when positive findings were reported, no precise response pattern could be identified in a reproducible way. In particular, results from HTST studies tend to exclude the role of a cell stressor for exposure to RF fields at nonthermal intensities. However, on the basis of lessons from microwave-assisted chemistry, we can assume that RF fields might affect heat-sensitive gene or protein expression to an extent larger than would be predicted from temperature change only. But in all likelihood, this would concern intensities higher than those relevant to usual human exposure. Conclusions The precise role of transcriptomics and proteomics in the screening of bioeffects from exposure to RF fields from mobile phones is still uncertain in view of the lack of positively identified phenotypic change and the lack of theoretical, as well as experimental, arguments for specific gene and/or protein response patterns after this kind of exposure. PMID:18795152

Proteomics screen to reveal molecular changes mediated by C722G missense mutation in CHRM2 gene.

PubMed

Hou, Dongyan; Chen, Ying; Liu, Jiamei; Xu, Lin; Zhang, Zhiyong; Zhang, Juan; Wang, Hua; Wang, Xin; Chen, Jin; Zhao, Rongrui; Hu, Aihua; Hakonarson, Hakon; Zhang, Lin; Shen, Yan

2013-08-26

Previously, we reported a missense mutation (C722G) in the M2-muscarinic acetylcholine receptor (CHRM2) gene associated with familial dilated cardiomyopathy. However, the exact molecular mechanisms by the related protein changes of CHRM2-C722G mutation induced are still unclear. CHRM2 and CHRM2-C722G lentiviral vector was infected to CHO cells. Proteomic analysis by label-free shotgun strategy and the STRING 9.0 software were performed. A total of 102 proteins with at least 2-fold change in the CHRM2-C722G group were identified, 42 proteins were up-regulated, whereas 57 were down-regulated. These altered proteins belong to three broad functional categories: (i) metabolic (e.g. Cytosolic acyl coenzyme A thioester hydrolase, Malate dehydrogenase); (ii) cytoskeletal (e.g. Actin-related protein, Myosin light polypeptide 6 and Alpha-actinin-1) and (iii) stress response (e.g. heat shock protein 70, Ras-related protein Rab-10). Interestingly, the marked differences in the expression of selected eight proteins (change >4.0-fold), were connected with many proteins related to apoptosis and immune/inflammatory response such as: FOS, BAX, MYC, TP53 and IL6. This novel study demonstrated for the first time a full-scale screening of the proteomics research by CHRM2-C722G mutation and profiled 102 changed proteins, of which, eight might be critical in cardiac dysfunction for future mapping. It was a full-scale screening of the proteomics research by CHRM2-C722G mutation. These proteins might serve as valuable biomarkers that could predict the presence of a precursor field. These proteins might serve to further explore the pathophysiological mechanisms in familial DCM patients with C176W mutation. Copyright © 2013 Elsevier B.V. All rights reserved.

Effect of postharvest ultraviolet-C treatment on the proteome changes in fresh cut mango (Mangifera indica L. cv. Chokanan).

PubMed

George, Dominic Soloman; Razali, Zuliana; Santhirasegaram, Vicknesha; Somasundram, Chandran

2016-06-01

Postharvest treatments of fruits using techniques such as ultraviolet-C have been linked with maintenance of the fruit quality as well as shelf-life extension. However, the effects of this treatment on the quality of fruits on a proteomic level remain unclear. This study was conducted in order to understand the response of mango fruit to postharvest UV-C irradiation. Approximately 380 reproducible spots were detected following two-dimensional gel electrophoresis. Through gel analysis, 24 spots were observed to be differentially expressed in UV-C treated fruits and 20 were successfully identified via LCMS/MS. Postharvest UV-C treatment resulted in degradative effects on these identified proteins of which 40% were related to stress response, 45% to energy and metabolism and 15% to ripening and senescence. In addition, quality and shelf-life analysis of control and irradiated mangoes was evaluated. UV-C was found to be successful in retention of quality and extension of shelf-life up to 15 days. Furthermore, UV-C was also successful in increasing antioxidants (total flavonoid, reducing power and ABTS scavenging activity) in mangoes. This study provides an overview of the effects of UV-C treatment on the quality of mango on a proteomic level as well as the potential of this treatment in shelf-life extension of fresh-cut fruits. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

AT_CHLORO, a comprehensive chloroplast proteome database with subplastidial localization and curated information on envelope proteins.

PubMed

Ferro, Myriam; Brugière, Sabine; Salvi, Daniel; Seigneurin-Berny, Daphné; Court, Magali; Moyet, Lucas; Ramus, Claire; Miras, Stéphane; Mellal, Mourad; Le Gall, Sophie; Kieffer-Jaquinod, Sylvie; Bruley, Christophe; Garin, Jérôme; Joyard, Jacques; Masselon, Christophe; Rolland, Norbert

2010-06-01

Recent advances in the proteomics field have allowed a series of high throughput experiments to be conducted on chloroplast samples, and the data are available in several public databases. However, the accurate localization of many chloroplast proteins often remains hypothetical. This is especially true for envelope proteins. We went a step further into the knowledge of the chloroplast proteome by focusing, in the same set of experiments, on the localization of proteins in the stroma, the thylakoids, and envelope membranes. LC-MS/MS-based analyses first allowed building the AT_CHLORO database (http://www.grenoble.prabi.fr/protehome/grenoble-plant-proteomics/), a comprehensive repertoire of the 1323 proteins, identified by 10,654 unique peptide sequences, present in highly purified chloroplasts and their subfractions prepared from Arabidopsis thaliana leaves. This database also provides extensive proteomics information (peptide sequences and molecular weight, chromatographic retention times, MS/MS spectra, and spectral count) for a unique chloroplast protein accurate mass and time tag database gathering identified peptides with their respective and precise analytical coordinates, molecular weight, and retention time. We assessed the partitioning of each protein in the three chloroplast compartments by using a semiquantitative proteomics approach (spectral count). These data together with an in-depth investigation of the literature were compiled to provide accurate subplastidial localization of previously known and newly identified proteins. A unique knowledge base containing extensive information on the proteins identified in envelope fractions was thus obtained, allowing new insights into this membrane system to be revealed. Altogether, the data we obtained provide unexpected information about plastidial or subplastidial localization of some proteins that were not suspected to be associated to this membrane system. The spectral counting-based strategy was further validated as the compartmentation of well known pathways (for instance, photosynthesis and amino acid, fatty acid, or glycerolipid biosynthesis) within chloroplasts could be dissected. It also allowed revisiting the compartmentation of the chloroplast metabolism and functions.

Gene duplications are extensive and contribute significantly to the toxic proteome of nematocysts isolated from Acropora digitifera (Cnidaria: Anthozoa: Scleractinia).

PubMed

Gacesa, Ranko; Chung, Ray; Dunn, Simon R; Weston, Andrew J; Jaimes-Becerra, Adrian; Marques, Antonio C; Morandini, André C; Hranueli, Daslav; Starcevic, Antonio; Ward, Malcolm; Long, Paul F

2015-10-13

Gene duplication followed by adaptive selection is a well-accepted process leading to toxin diversification in venoms. However, emergent genomic, transcriptomic and proteomic evidence now challenges this role to be at best equivocal to other processess . Cnidaria are arguably the most ancient phylum of the extant metazoa that are venomous and such provide a definitive ancestral anchor to examine the evolution of this trait. Here we compare predicted toxins from the translated genome of the coral Acropora digitifera to putative toxins revealed by proteomic analysis of soluble proteins discharged from nematocysts, to determine the extent to which gene duplications contribute to venom innovation in this reef-building coral species. A new bioinformatics tool called HHCompare was developed to detect potential gene duplications in the genomic data, which is made freely available ( https://github.com/rgacesa/HHCompare ). A total of 55 potential toxin encoding genes could be predicted from the A. digitifera genome, of which 36 (65 %) had likely arisen by gene duplication as evinced using the HHCompare tool and verified using two standard phylogeny methods. Surprisingly, only 22 % (12/55) of the potential toxin repertoire could be detected following rigorous proteomic analysis, for which only half (6/12) of the toxin proteome could be accounted for as peptides encoded by the gene duplicates. Biological activities of these toxins are dominatedby putative phospholipases and toxic peptidases. Gene expansions in A. digitifera venom are the most extensive yet described in any venomous animal, and gene duplication plays a significant role leading to toxin diversification in this coral species. Since such low numbers of toxins were detected in the proteome, it is unlikely that the venom is evolving rapidly by prey-driven positive natural selection. Rather we contend that the venom has a defensive role deterring predation or harm from interspecific competition and overgrowth by fouling organisms. Factors influencing translation of toxin encoding genes perhaps warrants more profound experimental consideration.

Proteomics to assess the role of phenotypic plasticity in aquatic organisms exposed to pollution and global warming.

PubMed

Silvestre, Frédéric; Gillardin, Virginie; Dorts, Jennifer

2012-11-01

Nowadays, the unprecedented rates of anthropogenic changes in ecosystems suggest that organisms have to migrate to new distributional ranges or to adapt commensurately quickly to new conditions to avoid becoming extinct. Pollution and global warming are two of the most important threats aquatic organisms will have to face in the near future. If genetic changes in a population in response to natural selection are extensively studied, the role of acclimation through phenotypic plasticity (the property of a given genotype to produce different phenotypes in response to particular environmental conditions) in a species to deal with new environmental conditions remains largely unknown. Proteomics is the extensive study of the protein complement of a genome. It is dynamic and depends on the specific tissue, developmental stage, and environmental conditions. As the final product of gene expression, it is subjected to several regulatory steps from gene transcription to the functional protein. Consequently, there is a discrepancy between the abundance of mRNA and the abundance of the corresponding protein. Moreover, proteomics is closer to physiology and gives a more functional knowledge of the regulation of gene expression than does transcriptomics. The study of protein-expression profiles, however, gives a better portrayal of the cellular phenotype and is considered as a key link between the genotype and the organismal phenotype. Under new environmental conditions, we can observe a shift of the protein-expression pattern defining a new cellular phenotype that can possibly improve the fitness of the organism. It is now necessary to define a proteomic norm of reaction for organisms acclimating to environmental stressors. Its link to fitness will give new insights into how organisms can evolve in a changing environment. The proteomic literature bearing on chronic exposure to pollutants and on acclimation to heat stress in aquatic organisms, as well as potential application of proteomics in evolutionary issues, are outlined. While the transcriptome responses are commonly investigated, proteomics approaches now need to be intensified, with the new perspective of integrating the cellular phenotype with the organismal phenotype and with the mechanisms of the regulation of gene expression, such as epigenetics.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Elzek, Mohamed A.; Rodland, Karin D.

In the past decade, there has been an increasing interest in applying proteomics to assist in understanding the pathogenesis of ovarian cancer, elucidating the mechanism of drug resistance, and in the development of biomarkers for early detection of ovarian cancer. Although ovarian cancer is a spectrum of different diseases, the strategies for diagnosis and treatment with surgery and adjuvant therapy are similar across ovarian cancer types, increasing the general applicability of discoveries made through proteomics research. While proteomic experiments face many difficulties which slow the pace of clinical applications, recent advances in proteomic technology contribute significantly to the identification ofmore » aberrant proteins and networks which can serve as targets for biomarker development and individualized therapies. This review provides a summary of the literature on proteomics’ contributions to ovarian cancer research and highlights the current issues, future directions, and challenges. In conclusion, we propose that protein-level characterization of primary lesion in ovarian cancer can decipher the mystery of this disease, improve diagnostic tools, and lead to more effective screening programs.« less

The proteomic landscape of triple-negative breast cancer.

PubMed

Lawrence, Robert T; Perez, Elizabeth M; Hernández, Daniel; Miller, Chris P; Haas, Kelsey M; Irie, Hanna Y; Lee, Su-In; Blau, C Anthony; Villén, Judit

2015-04-28

Triple-negative breast cancer is a heterogeneous disease characterized by poor clinical outcomes and a shortage of targeted treatment options. To discover molecular features of triple-negative breast cancer, we performed quantitative proteomics analysis of twenty human-derived breast cell lines and four primary breast tumors to a depth of more than 12,000 distinct proteins. We used this data to identify breast cancer subtypes at the protein level and demonstrate the precise quantification of biomarkers, signaling proteins, and biological pathways by mass spectrometry. We integrated proteomics data with exome sequence resources to identify genomic aberrations that affect protein expression. We performed a high-throughput drug screen to identify protein markers of drug sensitivity and understand the mechanisms of drug resistance. The genome and proteome provide complementary information that, when combined, yield a powerful engine for therapeutic discovery. This resource is available to the cancer research community to catalyze further analysis and investigation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Proteomic studies in zebrafish liver cells exposed to the brominated flame retardants HBCD and TBBPA.

PubMed

Kling, Peter; Förlin, Lars

2009-10-01

Proteomic effect screening in zebrafish liver cells was performed to generate hypotheses regarding single and mixed exposure to the BFRs HBCD and TBBPA. Responses at sublethal exposure were analysed by two-dimensional gel electrophoresis followed by MALDI-TOF and FT-ICR protein identification. Mixing of HBCD and TBBPA at sublethal doses of individual substances seemed to increase toxicity. Proteomic analyses revealed distinct exposure-specific and overlapping responses suggesting novel mechanisms with regard to HBCD and TBBPA exposure. While distinct HBCD responses were related to decreased protein metabolism, TBBPA revealed effects related to protein folding and NADPH production. Overlapping responses suggest increased gluconeogenesis (GAPDH and aldolase) while distinct mixture effects suggest a pronounced NADPH production and changes in proteins related to cell cycle control (prohibitin and crk-like oncogene). We conclude that mixtures containing HBCD and TBBPA may result in unexpected effects highlighting proteomics as a sensitive tool for detecting and hypothesis generation of mixture effects.

Role of the visual experience-dependent nascent proteome in neuronal plasticity

PubMed Central

Liu, Han-Hsuan; McClatchy, Daniel B; Schiapparelli, Lucio; Shen, Wanhua; Yates, John R

2018-01-01

Experience-dependent synaptic plasticity refines brain circuits during development. To identify novel protein synthesis-dependent mechanisms contributing to experience-dependent plasticity, we conducted a quantitative proteomic screen of the nascent proteome in response to visual experience in Xenopus optic tectum using bio-orthogonal metabolic labeling (BONCAT). We identified 83 differentially synthesized candidate plasticity proteins (CPPs). The CPPs form strongly interconnected networks and are annotated to a variety of biological functions, including RNA splicing, protein translation, and chromatin remodeling. Functional analysis of select CPPs revealed the requirement for eukaryotic initiation factor three subunit A (eIF3A), fused in sarcoma (FUS), and ribosomal protein s17 (RPS17) in experience-dependent structural plasticity in tectal neurons and behavioral plasticity in tadpoles. These results demonstrate that the nascent proteome is dynamic in response to visual experience and that de novo synthesis of machinery that regulates RNA splicing and protein translation is required for experience-dependent plasticity. PMID:29412139

Ultraweak photon emission and proteomics analyses in soybean under abiotic stress.

PubMed

Komatsu, Setsuko; Kamal, Abu Hena Mostafa; Makino, Takahiro; Hossain, Zahed

2014-07-01

Biophotons are ultraweak photon emissions that are closely related to various biological activities and processes. In mammals, biophoton emissions originate from oxidative bursts in immunocytes during immunological responses. Biophotons emitted from plant organs provide novel information about the physiological state of plant under in vivo condition. In this review, the principles and recent advances in the measurement of biophoton emissions in plants are described. Furthermore, examples of biophoton emission and proteomics in soybean under abiotic stress are reviewed and discussed. Finally, this review suggests that the application of proteomics should provide a better interpretation of plant response to biophoton emission and allow the identification of genes that will allow the screening of crops able to produce maximal yields, even in stressful environments. Copyright © 2014 Elsevier B.V. All rights reserved.

Label-free proteomic analysis to confirm the predicted proteome of Corynebacterium pseudotuberculosis under nitrosative stress mediated by nitric oxide.

PubMed

Silva, Wanderson M; Carvalho, Rodrigo D; Soares, Siomar C; Bastos, Isabela Fs; Folador, Edson L; Souza, Gustavo Hmf; Le Loir, Yves; Miyoshi, Anderson; Silva, Artur; Azevedo, Vasco

2014-12-04

Corynebacterium pseudotuberculosis biovar ovis is a facultative intracellular pathogen, and the etiological agent of caseous lymphadenitis in small ruminants. During the infection process, the bacterium is subjected to several stress conditions, including nitrosative stress, which is caused by nitric oxide (NO). In silico analysis of the genome of C. pseudotuberculosis ovis 1002 predicted several genes that could influence the resistance of this pathogen to nitrosative stress. Here, we applied high-throughput proteomics using high definition mass spectrometry to characterize the functional genome of C. pseudotuberculosis ovis 1002 in the presence of NO-donor Diethylenetriamine/nitric oxide adduct (DETA/NO), with the aim of identifying proteins involved in nitrosative stress resistance. We characterized 835 proteins, representing approximately 41% of the predicted proteome of C. pseudotuberculosis ovis 1002, following exposure to nitrosative stress. In total, 102 proteins were exclusive to the proteome of DETA/NO-induced cells, and a further 58 proteins were differentially regulated between the DETA/NO and control conditions. An interactomic analysis of the differential proteome of C. pseudotuberculosis in response to nitrosative stress was also performed. Our proteomic data set suggested the activation of both a general stress response and a specific nitrosative stress response, as well as changes in proteins involved in cellular metabolism, detoxification, transcriptional regulation, and DNA synthesis and repair. Our proteomic analysis validated previously-determined in silico data for C. pseudotuberculosis ovis 1002. In addition, proteomic screening performed in the presence of NO enabled the identification of a set of factors that can influence the resistance and survival of C. pseudotuberculosis during exposure to nitrosative stress.

Sex-Specific Biology of the Human Malaria Parasite Revealed from the Proteomes of Mature Male and Female Gametocytes.

PubMed

Miao, Jun; Chen, Zhao; Wang, Zenglei; Shrestha, Sony; Li, Xiaolian; Li, Runze; Cui, Liwang

2017-04-01

The gametocytes of the malaria parasites are obligate for perpetuating the parasite's life cycle through mosquitoes, but the sex-specific biology of gametocytes is poorly understood. We generated a transgenic line in the human malaria parasite Plasmodium falciparum , which allowed us to accurately separate male and female gametocytes by flow cytometry. In-depth analysis of the proteomes by liquid chromatography-tandem mass spectrometry identified 1244 and 1387 proteins in mature male and female gametocytes, respectively. GFP-tagging of nine selected proteins confirmed their sex-partitions to be agreeable with the results from the proteomic analysis. The sex-specific proteomes showed significant differences that are consistent with the divergent functions of the two sexes. Although the male-specific proteome (119 proteins) is enriched in proteins associated with the flagella and genome replication, the female-specific proteome (262 proteins) is more abundant in proteins involved in metabolism, translation and organellar functions. Compared with the Plasmodium berghei sex-specific proteomes, this study revealed both extensive conservation and considerable divergence between these two species, which reflect the disparities between the two species in proteins involved in cytoskeleton, lipid metabolism and protein degradation. Comparison with three sex-specific proteomes allowed us to obtain high-confidence lists of 73 and 89 core male- and female-specific/biased proteins conserved in Plasmodium The identification of sex-specific/biased proteomes in Plasmodium lays a solid foundation for understanding the molecular mechanisms underlying the unique sex-specific biology in this early-branching eukaryote. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Sex-Specific Biology of the Human Malaria Parasite Revealed from the Proteomes of Mature Male and Female Gametocytes *

PubMed Central

Miao, Jun; Chen, Zhao; Wang, Zenglei; Shrestha, Sony; Li, Xiaolian; Li, Runze; Cui, Liwang

2017-01-01

The gametocytes of the malaria parasites are obligate for perpetuating the parasite's life cycle through mosquitoes, but the sex-specific biology of gametocytes is poorly understood. We generated a transgenic line in the human malaria parasite Plasmodium falciparum, which allowed us to accurately separate male and female gametocytes by flow cytometry. In-depth analysis of the proteomes by liquid chromatography-tandem mass spectrometry identified 1244 and 1387 proteins in mature male and female gametocytes, respectively. GFP-tagging of nine selected proteins confirmed their sex-partitions to be agreeable with the results from the proteomic analysis. The sex-specific proteomes showed significant differences that are consistent with the divergent functions of the two sexes. Although the male-specific proteome (119 proteins) is enriched in proteins associated with the flagella and genome replication, the female-specific proteome (262 proteins) is more abundant in proteins involved in metabolism, translation and organellar functions. Compared with the Plasmodium berghei sex-specific proteomes, this study revealed both extensive conservation and considerable divergence between these two species, which reflect the disparities between the two species in proteins involved in cytoskeleton, lipid metabolism and protein degradation. Comparison with three sex-specific proteomes allowed us to obtain high-confidence lists of 73 and 89 core male- and female-specific/biased proteins conserved in Plasmodium. The identification of sex-specific/biased proteomes in Plasmodium lays a solid foundation for understanding the molecular mechanisms underlying the unique sex-specific biology in this early-branching eukaryote. PMID:28126901

A Rational Approach for Discovering and Validating Cancer Markers in Very Small Samples Using Mass Spectrometry and ELISA Microarrays

DOE PAGES

Zangar, Richard C.; Varnum, Susan M.; Covington, Chandice Y.; ...

2004-01-01

Identifying useful markers of cancer can be problematic due to limited amounts of sample. Some samples such as nipple aspirate fluid (NAF) or early-stage tumors are inherently small. Other samples such as serum are collected in larger volumes but archives of these samples are very valuable and only small amounts of each sample may be available for a single study. Also, given the diverse nature of cancer and the inherent variability in individual protein levels, it seems likely that the best approach to screen for cancer will be to determine the profile of a battery of proteins. As a result,more » a major challenge in identifying protein markers of disease is the ability to screen many proteins using very small amounts of sample. In this review, we outline some technological advances in proteomics that greatly advance this capability. Specifically, we propose a strategy for identifying markers of breast cancer in NAF that utilizes mass spectrometry (MS) to simultaneously screen hundreds or thousands of proteins in each sample. The best potential markers identified by the MS analysis can then be extensively characterized using an ELISA microarray assay. Because the microarray analysis is quantitative and large numbers of samples can be efficiently analyzed, this approach offers the ability to rapidly assess a battery of selected proteins in a manner that is directly relevant to traditional clinical assays.« less

The Succinated Proteome of FH-Mutant Tumours

PubMed Central

Yang, Ming; Ternette, Nicola; Su, Huizhong; Dabiri, Raliat; Kessler, Benedikt M.; Adam, Julie; Teh, Bin Tean; Pollard, Patrick J.

2014-01-01

Inherited mutations in the Krebs cycle enzyme fumarate hydratase (FH) predispose to hereditary leiomyomatosis and renal cell cancer (HLRCC). Loss of FH activity in HLRCC tumours causes accumulation of the Krebs cycle intermediate fumarate to high levels, which may act as an oncometabolite through various, but not necessarily mutually exclusive, mechanisms. One such mechanism, succination, is an irreversible non-enzymatic modification of cysteine residues by fumarate, to form S-(2-succino)cysteine (2SC). Previous studies have demonstrated that succination of proteins including glyceraldehyde 3-phosphate dehydrogenase (GAPDH), kelch-like ECH-associated protein 1 (KEAP1) and mitochondrial aconitase (ACO2) can have profound effects on cellular metabolism. Furthermore, immunostaining for 2SC is a sensitive and specific biomarker for HLRCC tumours. Here, we performed a proteomic screen on an FH-mutant tumour and two HLRCC-derived cancer cell lines and identified 60 proteins where one or more cysteine residues were succinated; 10 of which were succinated at cysteine residues either predicted, or experimentally proven, to be functionally significant. Bioinformatic enrichment analyses identified most succinated targets to be involved in redox signaling. To our knowledge, this is the first proteomic-based succination screen performed in human tumours and cancer-derived cells and has identified novel 2SC targets that may be relevant to the pathogenesis of HLRCC. PMID:25105836

Oxidative stress in aging: advances in proteomic approaches.

PubMed

Ortuño-Sahagún, Daniel; Pallàs, Mercè; Rojas-Mayorquín, Argelia E

2014-01-01

Aging is a gradual, complex process in which cells, tissues, organs, and the whole organism itself deteriorate in a progressive and irreversible manner that, in the majority of cases, implies pathological conditions that affect the individual's Quality of Life (QOL). Although extensive research efforts in recent years have been made, the anticipation of aging and prophylactic or treatment strategies continue to experience major limitations. In this review, the focus is essentially on the compilation of the advances generated by cellular expression profile analysis through proteomics studies (two-dimensional [2D] electrophoresis and mass spectrometry [MS]), which are currently used as an integral approach to study the aging process. Additionally, the relevance of the oxidative stress factors is discussed. Emphasis is placed on postmitotic tissues, such as neuronal, muscular, and red blood cells, which appear to be those most frequently studied with respect to aging. Additionally, models for the study of aging are discussed in a number of organisms, such as Caenorhabditis elegans, senescence-accelerated probe-8 mice (SAMP8), naked mole-rat (Heterocephalus glaber), and the beagle canine. Proteomic studies in specific tissues and organisms have revealed the extensive involvement of reactive oxygen species (ROS) and oxidative stress in aging.

Studies of a biochemical factory: tomato trichome deep expressed sequence tag sequencing and proteomics.

PubMed

Schilmiller, Anthony L; Miner, Dennis P; Larson, Matthew; McDowell, Eric; Gang, David R; Wilkerson, Curtis; Last, Robert L

2010-07-01

Shotgun proteomics analysis allows hundreds of proteins to be identified and quantified from a single sample at relatively low cost. Extensive DNA sequence information is a prerequisite for shotgun proteomics, and it is ideal to have sequence for the organism being studied rather than from related species or accessions. While this requirement has limited the set of organisms that are candidates for this approach, next generation sequencing technologies make it feasible to obtain deep DNA sequence coverage from any organism. As part of our studies of specialized (secondary) metabolism in tomato (Solanum lycopersicum) trichomes, 454 sequencing of cDNA was combined with shotgun proteomics analyses to obtain in-depth profiles of genes and proteins expressed in leaf and stem glandular trichomes of 3-week-old plants. The expressed sequence tag and proteomics data sets combined with metabolite analysis led to the discovery and characterization of a sesquiterpene synthase that produces beta-caryophyllene and alpha-humulene from E,E-farnesyl diphosphate in trichomes of leaf but not of stem. This analysis demonstrates the utility of combining high-throughput cDNA sequencing with proteomics experiments in a target tissue. These data can be used for dissection of other biochemical processes in these specialized epidermal cells.

Studies of a Biochemical Factory: Tomato Trichome Deep Expressed Sequence Tag Sequencing and Proteomics1[W][OA

PubMed Central

Schilmiller, Anthony L.; Miner, Dennis P.; Larson, Matthew; McDowell, Eric; Gang, David R.; Wilkerson, Curtis; Last, Robert L.

2010-01-01

Shotgun proteomics analysis allows hundreds of proteins to be identified and quantified from a single sample at relatively low cost. Extensive DNA sequence information is a prerequisite for shotgun proteomics, and it is ideal to have sequence for the organism being studied rather than from related species or accessions. While this requirement has limited the set of organisms that are candidates for this approach, next generation sequencing technologies make it feasible to obtain deep DNA sequence coverage from any organism. As part of our studies of specialized (secondary) metabolism in tomato (Solanum lycopersicum) trichomes, 454 sequencing of cDNA was combined with shotgun proteomics analyses to obtain in-depth profiles of genes and proteins expressed in leaf and stem glandular trichomes of 3-week-old plants. The expressed sequence tag and proteomics data sets combined with metabolite analysis led to the discovery and characterization of a sesquiterpene synthase that produces β-caryophyllene and α-humulene from E,E-farnesyl diphosphate in trichomes of leaf but not of stem. This analysis demonstrates the utility of combining high-throughput cDNA sequencing with proteomics experiments in a target tissue. These data can be used for dissection of other biochemical processes in these specialized epidermal cells. PMID:20431087

Proteome screening of pleural effusions identifies galectin 1 as a diagnostic biomarker and highlights several prognostic biomarkers for malignant mesothelioma.

PubMed

Mundt, Filip; Johansson, Henrik J; Forshed, Jenny; Arslan, Sertaç; Metintas, Muzaffer; Dobra, Katalin; Lehtiö, Janne; Hjerpe, Anders

2014-03-01

Malignant mesothelioma is an aggressive asbestos-induced cancer, and affected patients have a median survival of approximately one year after diagnosis. It is often difficult to reach a conclusive diagnosis, and ancillary measurements of soluble biomarkers could increase diagnostic accuracy. Unfortunately, few soluble mesothelioma biomarkers are suitable for clinical application. Here we screened the effusion proteomes of mesothelioma and lung adenocarcinoma patients to identify novel soluble mesothelioma biomarkers. We performed quantitative mass-spectrometry-based proteomics using isobaric tags for quantification and used narrow-range immobilized pH gradient/high-resolution isoelectric focusing (pH 4-4.25) prior to analysis by means of nano liquid chromatography coupled to MS/MS. More than 1,300 proteins were identified in pleural effusions from patients with malignant mesothelioma (n = 6), lung adenocarcinoma (n = 6), or benign mesotheliosis (n = 7). Data are available via ProteomeXchange with identifier PXD000531. The identified proteins included a set of known mesothelioma markers and proteins that regulate hallmarks of cancer such as invasion, angiogenesis, and immune evasion, plus several new candidate proteins. Seven candidates (aldo-keto reductase 1B10, apolipoprotein C-I, galectin 1, myosin-VIIb, superoxide dismutase 2, tenascin C, and thrombospondin 1) were validated by enzyme-linked immunosorbent assays in a larger group of patients with mesothelioma (n = 37) or metastatic carcinomas (n = 25) and in effusions from patients with benign, reactive conditions (n = 16). Galectin 1 was identified as overexpressed in effusions from lung adenocarcinoma relative to mesothelioma and was validated as an excellent predictor for metastatic carcinomas against malignant mesothelioma. Galectin 1, aldo-keto reductase 1B10, and apolipoprotein C-I were all identified as potential prognostic biomarkers for malignant mesothelioma. This analysis of the effusion proteome furthers our understanding of malignant mesothelioma, identified galectin 1 as a potential diagnostic biomarker, and highlighted several possible prognostic biomarkers of this disease.

Metabolomics-Driven Discovery of a Prenylated Isatin Antibiotic Produced by Streptomyces Species MBT28.

PubMed

Wu, Changsheng; Du, Chao; Gubbens, Jacob; Choi, Young Hae; van Wezel, Gilles P

2015-10-23

Actinomycetes are a major source of antimicrobials, anticancer compounds, and other medically important products, and their genomes harbor extensive biosynthetic potential. Major challenges in the screening of these microorganisms are to activate the expression of cryptic biosynthetic gene clusters and the development of technologies for efficient dereplication of known molecules. Here we report the identification of a previously unidentified isatin-type antibiotic produced by Streptomyces sp. MBT28, following a strategy based on NMR-based metabolomics combined with the introduction of streptomycin resistance in the producer strain. NMR-guided isolation by tracking the target proton signal resulted in the characterization of 7-prenylisatin (1) with antimicrobial activity against Bacillus subtilis. The metabolite-guided genome mining of Streptomyces sp. MBT28 combined with proteomics identified a gene cluster with an indole prenyltransferase that catalyzes the conversion of tryptophan into 7-prenylisatin. This study underlines the applicability of NMR-based metabolomics in facilitating the discovery of novel antibiotics.

Proteomic changes involved in tenderization of bovine Longissimus dorsi muscle during prolonged ageing.

PubMed

Polati, Rita; Menini, Michele; Robotti, Elisa; Millioni, Renato; Marengo, Emilio; Novelli, Enrico; Balzan, Stefania; Cecconi, Daniela

2012-12-01

To study proteomic changes involved in tenderization of bovine Longissimus dorsi four Charolaise heifers and four Charolaise bull's muscles were sampled at slaughter after early and long ageing (2-4°C for 12 and 26days respectively). Descriptive sensory evaluation of samples were performed and their tenderness evaluated by Warner-Bratzler shear force test. Protein composition of fresh muscle and of meat aged was analysed by cartesian and polar 2-D electrophoresis. Student's t-test and Ranking-PCA analyses were performed to detect proteomic modulation, and the selected protein spots were identified by nano-HPLC-Chip MS/MS. This research has demonstrated that there are no differences between proteomic patterns of male and females Longissimus dorsi muscle, and that the extension of ageing beyond 12days, did not brings any concrete advantage in terms of sensory quality. Furthermore, the data presented here demonstrated that meat maturation caused changes of the abundance of proteins involved in metabolic, structural, and stress related processes. Copyright © 2012 Elsevier Ltd. All rights reserved.

Brucella proteomes--a review.

PubMed

DelVecchio, Vito G; Wagner, Mary Ann; Eschenbrenner, Michel; Horn, Troy A; Kraycer, Jo Ann; Estock, Frank; Elzer, Phil; Mujer, Cesar V

2002-12-20

The proteomes of selected Brucella spp. have been extensively analyzed by utilizing current proteomic technology involving 2-DE and MALDI-MS. In Brucella melitensis, more than 500 proteins were identified. The rapid and large-scale identification of proteins in this organism was accomplished by using the annotated B. melitensis genome which is now available in the GenBank. Coupled with new and powerful tools for data analysis, differentially expressed proteins were identified and categorized into several classes. A global overview of protein expression patterns emerged, thereby facilitating the simultaneous analysis of different metabolic pathways in B. melitensis. Such a global characterization would not have been possible by using time consuming and traditional biochemical approaches. The era of post-genomic technology offers new and exciting opportunities to understand the complete biology of different Brucella species.

Integration of gel-based proteome data with pProRep.

PubMed

Laukens, Kris; Matthiesen, Rune; Lemière, Filip; Esmans, Eddy; Onckelen, Harry Van; Jensen, Ole Nørregaard; Witters, Erwin

2006-11-15

pProRep is a web application integrating electrophoretic and mass spectral data from proteome analyses into a relational database. The graphical web-interface allows users to upload, analyse and share experimental proteome data. It offers researchers the possibility to query all previously analysed datasets and can visualize selected features, such as the presence of a certain set of ions in a peptide mass spectrum, on the level of the two-dimensional gel. The pProRep package and instructions for its use can be downloaded from http://www.ptools.ua.ac.be/pProRep. The application requires a web server that runs PHP 5 (http://www.php.net) and MySQL. Some (non-essential) extensions need additional freely available libraries: details are described in the installation instructions.

Intact mass detection, interpretation, and visualization to automate Top-Down proteomics on a large scale

PubMed Central

Durbin, Kenneth R.; Tran, John C.; Zamdborg, Leonid; Sweet, Steve M. M.; Catherman, Adam D.; Lee, Ji Eun; Li, Mingxi; Kellie, John F.; Kelleher, Neil L.

2011-01-01

Applying high-throughput Top-Down MS to an entire proteome requires a yet-to-be-established model for data processing. Since Top-Down is becoming possible on a large scale, we report our latest software pipeline dedicated to capturing the full value of intact protein data in automated fashion. For intact mass detection, we combine algorithms for processing MS1 data from both isotopically resolved (FT) and charge-state resolved (ion trap) LC-MS data, which are then linked to their fragment ions for database searching using ProSight. Automated determination of human keratin and tubulin isoforms is one result. Optimized for the intricacies of whole proteins, new software modules visualize proteome-scale data based on the LC retention time and intensity of intact masses and enable selective detection of PTMs to automatically screen for acetylation, phosphorylation, and methylation. Software functionality was demonstrated using comparative LC-MS data from yeast strains in addition to human cells undergoing chemical stress. We further these advances as a key aspect of realizing Top-Down MS on a proteomic scale. PMID:20848673

Functional proteomics within the genus Lactobacillus.

PubMed

De Angelis, Maria; Calasso, Maria; Cavallo, Noemi; Di Cagno, Raffaella; Gobbetti, Marco

2016-03-01

Lactobacillus are mainly used for the manufacture of fermented dairy, sourdough, meat, and vegetable foods or used as probiotics. Under optimal processing conditions, Lactobacillus strains contribute to food functionality through their enzyme portfolio and the release of metabolites. An extensive genomic diversity analysis was conducted to elucidate the core features of the genus Lactobacillus, and to provide a better comprehension of niche adaptation of the strains. However, proteomics is an indispensable "omics" science to elucidate the proteome diversity, and the mechanisms of regulation and adaptation of Lactobacillus strains. This review focuses on the novel and comprehensive knowledge of functional proteomics and metaproteomics of Lactobacillus species. A large list of proteomic case studies of different Lactobacillus species is provided to illustrate the adaptability of the main metabolic pathways (e.g., carbohydrate transport and metabolism, pyruvate metabolism, proteolytic system, amino acid metabolism, and protein synthesis) to various life conditions. These investigations have highlighted that lactobacilli modulate the level of a complex panel of proteins to growth/survive in different ecological niches. In addition to the general regulation and stress response, specific metabolic pathways can be switched on and off, modifying the behavior of the strains. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparison of methods for profiling O-glycosylation: Human Proteome Organisation Human Disease Glycomics/Proteome Initiative multi-institutional study of IgA1.

PubMed

Wada, Yoshinao; Dell, Anne; Haslam, Stuart M; Tissot, Bérangère; Canis, Kévin; Azadi, Parastoo; Bäckström, Malin; Costello, Catherine E; Hansson, Gunnar C; Hiki, Yoshiyuki; Ishihara, Mayumi; Ito, Hiromi; Kakehi, Kazuaki; Karlsson, Niclas; Hayes, Catherine E; Kato, Koichi; Kawasaki, Nana; Khoo, Kay-Hooi; Kobayashi, Kunihiko; Kolarich, Daniel; Kondo, Akihiro; Lebrilla, Carlito; Nakano, Miyako; Narimatsu, Hisashi; Novak, Jan; Novotny, Milos V; Ohno, Erina; Packer, Nicolle H; Palaima, Elizabeth; Renfrow, Matthew B; Tajiri, Michiko; Thomsson, Kristina A; Yagi, Hirokazu; Yu, Shin-Yi; Taniguchi, Naoyuki

2010-04-01

The Human Proteome Organisation Human Disease Glycomics/Proteome Initiative recently coordinated a multi-institutional study that evaluated methodologies that are widely used for defining the N-glycan content in glycoproteins. The study convincingly endorsed mass spectrometry as the technique of choice for glycomic profiling in the discovery phase of diagnostic research. The present study reports the extension of the Human Disease Glycomics/Proteome Initiative's activities to an assessment of the methodologies currently used for O-glycan analysis. Three samples of IgA1 isolated from the serum of patients with multiple myeloma were distributed to 15 laboratories worldwide for O-glycomics analysis. A variety of mass spectrometric and chromatographic procedures representative of current methodologies were used. Similar to the previous N-glycan study, the results convincingly confirmed the pre-eminent performance of MS for O-glycan profiling. Two general strategies were found to give the most reliable data, namely direct MS analysis of mixtures of permethylated reduced glycans in the positive ion mode and analysis of native reduced glycans in the negative ion mode using LC-MS approaches. In addition, mass spectrometric methodologies to analyze O-glycopeptides were also successful.

Next-Generation Proteomics and Its Application to Clinical Breast Cancer Research.

PubMed

Mardamshina, Mariya; Geiger, Tamar

2017-10-01

Proteomics technology aims to map the protein landscapes of biological samples, and it can be applied to a variety of samples, including cells, tissues, and body fluids. Because the proteins are the main functional molecules in the cells, their levels reflect much more accurately the cellular phenotype and the regulatory processes within them than gene levels, mutations, and even mRNA levels. With the advancement in the technology, it is possible now to obtain comprehensive views of the biological systems and to study large patient cohorts in a streamlined manner. In this review we discuss the technological advancements in mass spectrometry-based proteomics, which allow analysis of breast cancer tissue samples, leading to the first large-scale breast cancer proteomics studies. Furthermore, we discuss the technological developments in blood-based biomarker discovery, which provide the basis for future development of assays for routine clinical use. Although these are only the first steps in implementation of proteomics into the clinic, extensive collaborative work between these worlds will undoubtedly lead to major discoveries and advances in clinical practice. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Screening Novel Molecular Targets of Metformin in Breast Cancer by Proteomic Approach

PubMed Central

Al-Zaidan, Lobna; El Ruz, Rasha Abu; Malki, Ahmed M.

2017-01-01

Metformin is a commonly prescribed antihyperglycemic drug, and has been investigated in vivo and in vitro for its effect to improve the comorbidity of diabetes and various types of cancers. Several studies investigated the therapeutic mechanisms of metformin on cancer cells, but the exact mechanism of metformin’s effect on the proteomic pathways of cancer cells is yet to be further investigated. The main objective of our research line is to discover safe and alternative therapeutic options for breast cancer, we aimed in this study to design a novel “bottom up proteomics workflow” in which proteins were first broken into peptides to reveal their identity, then the proteomes were precisely evaluated using spectrometry analysis. In our study, metformin suppressed cell proliferation and induced apoptosis in human breast carcinoma cell line MCF-7 with minimal toxicity to normal breast epithelial cells MCF-10. Metformin induced apoptosis by arresting cells in G1 phase as evaluated by flow cytometric analysis. Moreover, The G1 phase arrest for the MCF-7 has been confirmed by increased expression levels of p21 and reduction in cyclin D1 level. Additionally, metformin increased the expression levels of p53, Bax, Bad while it reduced expression levels of Akt, Bcl-2, and Mdm2. The study employed a serviceable strategy that investigates metformin-dependent changes in the proteome using a literature-derived network. The protein extracts of the treated and untreated cell lines were analyzed employing proteomic approaches; the findings conveyed a proposed mechanism of the effectual tactics of metformin on breast cancer cells. Metformin proposed an antibreast cancer effect through the examination of the proteomic pathways upon the MCF-7 and MCF-10A exposure to the drug. Our findings proposed prolific proteomic changes that revealed the therapeutic mechanisms of metformin on breast cancer cells upon their exposure. In conclusion, the reported proteomic pathways lead to increase the understanding of breast cancer prognosis and permit future studies to examine the effect of metformin on the proteomic pathways against other types of cancers. Finally, it suggests the possibility to develop further therapeutic generations of metformin with increased anticancer effect through targeting specific proteomes. PMID:29085821

Ultra-sensitive high performance liquid chromatography-laser-induced fluorescence based proteomics for clinical applications.

PubMed

Patil, Ajeetkumar; Bhat, Sujatha; Pai, Keerthilatha M; Rai, Lavanya; Kartha, V B; Chidangil, Santhosh

2015-09-08

An ultra-sensitive high performance liquid chromatography-laser induced fluorescence (HPLC-LIF) based technique has been developed by our group at Manipal, for screening, early detection, and staging for various cancers, using protein profiling of clinical samples like, body fluids, cellular specimens, and biopsy-tissue. More than 300 protein profiles of different clinical samples (serum, saliva, cellular samples and tissue homogenates) from volunteers (normal, and different pre-malignant/malignant conditions) were recorded using this set-up. The protein profiles were analyzed using principal component analysis (PCA) to achieve objective detection and classification of malignant, premalignant and healthy conditions with high sensitivity and specificity. The HPLC-LIF protein profiling combined with PCA, as a routine method for screening, diagnosis, and staging of cervical cancer and oral cancer, is discussed in this paper. In recent years, proteomics techniques have advanced tremendously in life sciences and medical sciences for the detection and identification of proteins in body fluids, tissue homogenates and cellular samples to understand biochemical mechanisms leading to different diseases. Some of the methods include techniques like high performance liquid chromatography, 2D-gel electrophoresis, MALDI-TOF-MS, SELDI-TOF-MS, CE-MS and LC-MS techniques. We have developed an ultra-sensitive high performance liquid chromatography-laser induced fluorescence (HPLC-LIF) based technique, for screening, early detection, and staging for various cancers, using protein profiling of clinical samples like, body fluids, cellular specimens, and biopsy-tissue. More than 300 protein profiles of different clinical samples (serum, saliva, cellular samples and tissue homogenates) from healthy and volunteers with different malignant conditions were recorded by using this set-up. The protein profile data were analyzed using principal component analysis (PCA) for objective classification and detection of malignant, premalignant and healthy conditions. The method is extremely sensitive to detect proteins with limit of detection of the order of femto-moles. The HPLC-LIF combined with PCA as a potential proteomic method for the diagnosis of oral cancer and cervical cancer has been discussed in this paper. This article is part of a Special Issue entitled: Proteomics in India. Copyright © 2015 Elsevier B.V. All rights reserved.

Statistical Methods for Proteomic Biomarker Discovery based on Feature Extraction or Functional Modeling Approaches.

PubMed

Morris, Jeffrey S

2012-01-01

In recent years, developments in molecular biotechnology have led to the increased promise of detecting and validating biomarkers, or molecular markers that relate to various biological or medical outcomes. Proteomics, the direct study of proteins in biological samples, plays an important role in the biomarker discovery process. These technologies produce complex, high dimensional functional and image data that present many analytical challenges that must be addressed properly for effective comparative proteomics studies that can yield potential biomarkers. Specific challenges include experimental design, preprocessing, feature extraction, and statistical analysis accounting for the inherent multiple testing issues. This paper reviews various computational aspects of comparative proteomic studies, and summarizes contributions I along with numerous collaborators have made. First, there is an overview of comparative proteomics technologies, followed by a discussion of important experimental design and preprocessing issues that must be considered before statistical analysis can be done. Next, the two key approaches to analyzing proteomics data, feature extraction and functional modeling, are described. Feature extraction involves detection and quantification of discrete features like peaks or spots that theoretically correspond to different proteins in the sample. After an overview of the feature extraction approach, specific methods for mass spectrometry ( Cromwell ) and 2D gel electrophoresis ( Pinnacle ) are described. The functional modeling approach involves modeling the proteomic data in their entirety as functions or images. A general discussion of the approach is followed by the presentation of a specific method that can be applied, wavelet-based functional mixed models, and its extensions. All methods are illustrated by application to two example proteomic data sets, one from mass spectrometry and one from 2D gel electrophoresis. While the specific methods presented are applied to two specific proteomic technologies, MALDI-TOF and 2D gel electrophoresis, these methods and the other principles discussed in the paper apply much more broadly to other expression proteomics technologies.

Proteomic and N-glycoproteomic quantification reveal aberrant changes in the human saliva of oral ulcer patients.

PubMed

Zhang, Ying; Wang, Xi; Cui, Dan; Zhu, Jun

2016-12-01

Human whole saliva is a vital body fluid for studying the physiology and pathology of the oral cavity. As a powerful technique for biomarker discovery, MS-based proteomic strategies have been introduced for saliva analysis and identified hundreds of proteins and N-glycosylation sites. However, there is still a lack of quantitative analysis, which is necessary for biomarker screening and biological research. In this study, we establish an integrated workflow by the combination of stable isotope dimethyl labeling, HILIC enrichment, and high resolution MS for both quantification of the global proteome and N-glycoproteome of human saliva from oral ulcer patients. With the help of advanced bioinformatics, we comprehensively studied oral ulcers at both protein and glycoprotein scales. Bioinformatics analyses revealed that starch digestion and protein degradation activities are inhibited while the immune response is promoted in oral ulcer saliva. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Detection of dysregulated protein-association networks by high-throughput proteomics predicts cancer vulnerabilities.

PubMed

Lapek, John D; Greninger, Patricia; Morris, Robert; Amzallag, Arnaud; Pruteanu-Malinici, Iulian; Benes, Cyril H; Haas, Wilhelm

2017-10-01

The formation of protein complexes and the co-regulation of the cellular concentrations of proteins are essential mechanisms for cellular signaling and for maintaining homeostasis. Here we use isobaric-labeling multiplexed proteomics to analyze protein co-regulation and show that this allows the identification of protein-protein associations with high accuracy. We apply this 'interactome mapping by high-throughput quantitative proteome analysis' (IMAHP) method to a panel of 41 breast cancer cell lines and show that deviations of the observed protein co-regulations in specific cell lines from the consensus network affects cellular fitness. Furthermore, these aberrant interactions serve as biomarkers that predict the drug sensitivity of cell lines in screens across 195 drugs. We expect that IMAHP can be broadly used to gain insight into how changing landscapes of protein-protein associations affect the phenotype of biological systems.

Clostridium perfringens bacteriophages FCP39O and FCP26F: genomic organization and proteomic analysis of the virions

USDA-ARS?s Scientific Manuscript database

Initial screening for bacteriophages lytic for Clostridium perfringens was performed utilizing filtered samples obtained from poultry (intestinal material), soil, sewage and poultry processing drainage water. Lytic phage preparations were initially characterized by transmission electron microscopy ...

Matrix metalloproteinase proteomics: substrates, targets, and therapy.

PubMed

Morrison, Charlotte J; Butler, Georgina S; Rodríguez, David; Overall, Christopher M

2009-10-01

Proteomics encompasses powerful techniques termed 'degradomics' for unbiased high-throughput protease substrate discovery screens that have been applied to an important family of extracellular proteases, the matrix metalloproteinases (MMPs). Together with the data generated from genetic deletion and transgenic mouse models and genomic profiling, these screens can uncover the diverse range of MMP functions, reveal which MMPs and MMP-mediated pathways exacerbate pathology, and which are involved in protection and the resolution of disease. This information can be used to identify and validate candidate drug targets and antitargets, and is critical for the development of new inhibitors of MMP function. Such inhibitors may target either the MMP directly in a specific manner or pathways upstream and downstream of MMP activity that are mediating deleterious effects in disease. Since MMPs do not operate alone but are part of the 'protease web', it is necessary to use system-wide approaches to understand MMP proteolysis in vivo, to discover new biological roles and their potential for therapeutic modification.

Proteomics analysis of human placenta reveals glutathione metabolism dysfunction as the underlying pathogenesis for preeclampsia.

PubMed

Jin, Xiaohan; Xu, Zhongwei; Cao, Jin; Shao, Ping; Zhou, Maobin; Qin, Zhe; Liu, Yan; Yu, Fang; Zhou, Xin; Ji, Wenjie; Cai, Wei; Ma, Yongqiang; Wang, Chengyan; Shan, Nana; Yang, Ning; Chen, Xu; Li, Yuming

2017-09-01

Hypertensive disorder in pregnancy (HDP) refers to a series of diseases that cause the hypertension during pregnancy, including HDP, preeclampsia (PE) and eclampsia. This study screens differentially expressed proteins of placenta tissues in PE cases using 2D LC-MS/MS quantitative proteomics strategy. A total of 2281 proteins are quantified, of these, 145 altering expression proteins are successfully screened between PE and control cases (p<0.05). Bioinformatics analysis suggests that these proteins are mainly involved in many biological processes, such as oxidation reduction, mitochondrion organization, and acute inflammatory response. Especially, the glutamine metabolic process related molecules, GPX1, GPX3, SMS, GGCT, GSTK1, NFκB, GSTT2, SOD1 and GCLM, are involved in the switching process from oxidized glutathione (GSSG) conversion to the reduced glutathione (GSH) by glutathione, mercapturic acid and arginine metabolism process. Results of this study revealed that glutathione metabolism disorder of placenta tissues may contribute to the occurrence of PE disease. Copyright © 2017. Published by Elsevier B.V.

Identification of Autophagosome-associated Proteins and Regulators by Quantitative Proteomic Analysis and Genetic Screens*

PubMed Central

Dengjel, Jörn; Høyer-Hansen, Maria; Nielsen, Maria O.; Eisenberg, Tobias; Harder, Lea M.; Schandorff, Søren; Farkas, Thomas; Kirkegaard, Thomas; Becker, Andrea C.; Schroeder, Sabrina; Vanselow, Katja; Lundberg, Emma; Nielsen, Mogens M.; Kristensen, Anders R.; Akimov, Vyacheslav; Bunkenborg, Jakob; Madeo, Frank; Jäättelä, Marja; Andersen, Jens S.

2012-01-01

Autophagy is one of the major intracellular catabolic pathways, but little is known about the composition of autophagosomes. To study the associated proteins, we isolated autophagosomes from human breast cancer cells using two different biochemical methods and three stimulus types: amino acid deprivation or rapamycin or concanamycin A treatment. The autophagosome-associated proteins were dependent on stimulus, but a core set of proteins was stimulus-independent. Remarkably, proteasomal proteins were abundant among the stimulus-independent common autophagosome-associated proteins, and the activation of autophagy significantly decreased the cellular proteasome level and activity supporting interplay between the two degradation pathways. A screen of yeast strains defective in the orthologs of the human genes encoding for a common set of autophagosome-associated proteins revealed several regulators of autophagy, including subunits of the retromer complex. The combined spatiotemporal proteomic and genetic data sets presented here provide a basis for further characterization of autophagosome biogenesis and cargo selection. PMID:22311637

The Impact of the Glomerular Filtration Rate on the Human Plasma Proteome.

PubMed

Christensson, Anders; Ash, Jessica A; DeLisle, Robert K; Gaspar, Fraser W; Ostroff, Rachel; Grubb, Anders; Lindström, Veronica; Bruun, Laila; Williams, Steve A

2018-05-01

The application of proteomics in chronic kidney disease (CKD) can potentially uncover biomarkers and pathways that are predictive of disease. Within this context, this study examines the relationship between the human plasma proteome and glomerular filtration rate (GFR) as measured by iohexol clearance in a cohort from Sweden (n = 389; GFR range: 8-100 mL min -1 /1.73 m 2 ). A total of 2893 proteins are quantified using a modified aptamer assay. A large proportion of the proteome is associated with GFR, reinforcing the concept that CKD affects multiple physiological systems (individual protein-GFR correlations listed here). Of these, cystatin C shows the most significant correlation with GFR (rho = -0.85, p = 1.2 × 10 -97 ), establishing strong validation for the use of this biomarker in CKD diagnostics. Among the other highly significant protein markers are insulin-like growth factor-binding protein 6, neuroblastoma suppressor of tumorigenicity 1, follistatin-related protein 3, trefoil factor 3, and beta-2 microglobulin. These proteins may indicate an imbalance in homeostasis across a variety of cellular processes, which may be underlying renal dysfunction. Overall, this study represents the most extensive characterization of the plasma proteome and its relation to GFR to date, and suggests the diagnostic and prognostic value of proteomics for CKD across all stages. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Advances in targeted proteomics and applications to biomedical research

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Tujin; Song, Ehwang; Nie, Song

Targeted proteomics technique has emerged as a powerful protein quantification tool in systems biology, biomedical research, and increasing for clinical applications. The most widely used targeted proteomics approach, selected reaction monitoring (SRM), also known as multiple reaction monitoring (MRM), can be used for quantification of cellular signaling networks and preclinical verification of candidate protein biomarkers. As an extension to our previous review on advances in SRM sensitivity (Shi et al., Proteomics, 12, 1074–1092, 2012) herein we review recent advances in the method and technology for further enhancing SRM sensitivity (from 2012 to present), and highlighting its broad biomedical applications inmore » human bodily fluids, tissue and cell lines. Furthermore, we also review two recently introduced targeted proteomics approaches, parallel reaction monitoring (PRM) and data-independent acquisition (DIA) with targeted data extraction on fast scanning high-resolution accurate-mass (HR/AM) instruments. Such HR/AM targeted quantification with monitoring all target product ions addresses SRM limitations effectively in specificity and multiplexing; whereas when compared to SRM, PRM and DIA are still in the infancy with a limited number of applications. Thus, for HR/AM targeted quantification we focus our discussion on method development, data processing and analysis, and its advantages and limitations in targeted proteomics. Finally, general perspectives on the potential of achieving both high sensitivity and high sample throughput for large-scale quantification of hundreds of target proteins are discussed.« less

The Brain Proteome of the Ubiquitin Ligase Peli1 Knock-Out Mouse during Experimental Autoimmune Encephalomyelitis.

PubMed

Lereim, Ragnhild Reehorst; Oveland, Eystein; Xiao, Yichuan; Torkildsen, Øivind; Wergeland, Stig; Myhr, Kjell-Morten; Sun, Shao-Cong; Berven, Frode S

2016-09-01

The ubiquitin ligase Peli1 has previously been suggested as a potential treatment target in multiple sclerosis. In the multiple sclerosis disease model, experimental autoimmune encephalomyelitis, Peli1 knock-out led to less activated microglia and less inflammation in the central nervous system. Despite being important in microglia, Peli1 expression has also been detected in glial and neuronal cells. In the present study the overall brain proteomes of Peli1 knock-out mice and wild-type mice were compared prior to experimental autoimmune encephalomyelitis induction, at onset of the disease and at disease peak. Brain samples from the frontal hemisphere, peripheral from the extensive inflammatory foci, were analyzed using TMT-labeling of sample pools, and the discovered proteins were verified in individual mice using label-free proteomics. The greatest proteomic differences between Peli1 knock-out and wild-type mice were observed at the disease peak. In Peli1 knock-out a higher degree of antigen presentation, increased activity of adaptive and innate immune cells and alterations to proteins involved in iron metabolism were observed during experimental autoimmune encephalomyelitis. These results unravel global effects to the brain proteome when abrogating Peli1 expression, underlining the importance of Peli1 as a regulator of the immune response also peripheral to inflammatory foci during experimental autoimmune encephalomyelitis. The proteomics data is available in PRIDE with accession PXD003710.

Proteome analysis of a hepatocyte-specific BIRC5 (survivin)-knockout mouse model during liver regeneration.

PubMed

Bracht, Thilo; Hagemann, Sascha; Loscha, Marius; Megger, Dominik A; Padden, Juliet; Eisenacher, Martin; Kuhlmann, Katja; Meyer, Helmut E; Baba, Hideo A; Sitek, Barbara

2014-06-06

The Baculoviral IAP repeat-containing protein 5 (BIRC5), also known as inhibitor of apoptosis protein survivin, is a member of the chromosomal passenger complex and a key player in mitosis. To investigate the function of BIRC5 in liver regeneration, we analyzed a hepatocyte-specific BIRC5-knockout mouse model using a quantitative label-free proteomics approach. Here, we present the analyses of the proteome changes in hepatocyte-specific BIRC5-knockout mice compared to wildtype mice, as well as proteome changes during liver regeneration induced by partial hepatectomy in wildtype mice and mice lacking hepatic BIRC5, respectively. The BIRC5-knockout mice showed an extensive overexpression of proteins related to cellular maintenance, organization and protein synthesis. Key regulators of cell growth, transcription and translation MTOR and STAT1/STAT2 were found to be overexpressed. During liver regeneration proteome changes representing a response to the mitotic stimulus were detected in wildtype mice. Mainly proteins corresponding to proliferation, cell cycle and cytokinesis were up-regulated. The hepatocyte-specific BIRC5-knockout mice showed impaired liver regeneration, which had severe consequences on the proteome level. However, several proteins with function in mitosis were found to be up-regulated upon the proliferative stimulus. Our results show that the E3 ubiquitin-protein ligase UHRF1 is strongly up-regulated during liver regeneration independently of BIRC5.

Proteomics: A valuable approach to elucidate spermatozoa post -testicular maturation in the endangered Acipenseridae family.

PubMed

Boccaletto, Pietro; Siddique, Mohammad Abdul Momin; Cosson, Jacky

2018-05-01

Proteomics techniques, such as two-dimensional polyacrylamide gel electrophoresis, mass spectrometry, and differential gel electrophoresis, have been extensively used to describe the protein composition of male gametes in different animals, mainly mammals. They have also provided a deeper understanding of protein functions involved in sperm processes, as in processes that in humans lead to male infertility. However, few studies focus on fish sperm proteomics and even fewer have tried to explore the proteomic profile of Sturgeon spermatozoa. Sturgeon is an endangered, ancient group of fish species exploited mostly for caviar. In this fish group, a part of the process that leads to final functional maturation of spermatozoa so as to have the capability to activate eggs during the fertilization process. This process has a broad similarity to post-testicular maturation in mammals; where spermatozoa leaving the testes must be mixed with seminal fluid along the transit through the Wolffian ducts to modify its surface membrane protein composition, leading to axonemal and acrosomal competence. The aim of this study was to review the current literature on various proteomic techniques, their usefulness in separating, identifying and studying the proteome composition of the fish spermatozoon, as well as their potential applications in studying the post-testicular maturation process in Sturgeon. Such understanding could lead to development of more sophisticated aquaculture techniques, favorable for sturgeon reproduction. Copyright © 2018 Elsevier B.V. All rights reserved.

Chemical proteomics approaches for identifying the cellular targets of natural products

PubMed Central

Sieber, S. A.

2016-01-01

Covering: 2010 up to 2016 Deconvoluting the mode of action of natural products and drugs remains one of the biggest challenges in chemistry and biology today. Chemical proteomics is a growing area of chemical biology that seeks to design small molecule probes to understand protein function. In the context of natural products, chemical proteomics can be used to identify the protein binding partners or targets of small molecules in live cells. Here, we highlight recent examples of chemical probes based on natural products and their application for target identification. The review focuses on probes that can be covalently linked to their target proteins (either via intrinsic chemical reactivity or via the introduction of photocrosslinkers), and can be applied “in situ” – in living systems rather than cell lysates. We also focus here on strategies that employ a click reaction, the copper-catalysed azide–alkyne cycloaddition reaction (CuAAC), to allow minimal functionalisation of natural product scaffolds with an alkyne or azide tag. We also discuss ‘competitive mode’ approaches that screen for natural products that compete with a well-characterised chemical probe for binding to a particular set of protein targets. Fuelled by advances in mass spectrometry instrumentation and bioinformatics, many modern strategies are now embracing quantitative proteomics to help define the true interacting partners of probes, and we highlight the opportunities this rapidly evolving technology provides in chemical proteomics. Finally, some of the limitations and challenges of chemical proteomics approaches are discussed. PMID:27098809

Chemical proteomics approaches for identifying the cellular targets of natural products.

PubMed

Wright, M H; Sieber, S A

2016-05-04

Covering: 2010 up to 2016Deconvoluting the mode of action of natural products and drugs remains one of the biggest challenges in chemistry and biology today. Chemical proteomics is a growing area of chemical biology that seeks to design small molecule probes to understand protein function. In the context of natural products, chemical proteomics can be used to identify the protein binding partners or targets of small molecules in live cells. Here, we highlight recent examples of chemical probes based on natural products and their application for target identification. The review focuses on probes that can be covalently linked to their target proteins (either via intrinsic chemical reactivity or via the introduction of photocrosslinkers), and can be applied "in situ" - in living systems rather than cell lysates. We also focus here on strategies that employ a click reaction, the copper-catalysed azide-alkyne cycloaddition reaction (CuAAC), to allow minimal functionalisation of natural product scaffolds with an alkyne or azide tag. We also discuss 'competitive mode' approaches that screen for natural products that compete with a well-characterised chemical probe for binding to a particular set of protein targets. Fuelled by advances in mass spectrometry instrumentation and bioinformatics, many modern strategies are now embracing quantitative proteomics to help define the true interacting partners of probes, and we highlight the opportunities this rapidly evolving technology provides in chemical proteomics. Finally, some of the limitations and challenges of chemical proteomics approaches are discussed.

Screening trematodes for novel intervention targets: a proteomic and immunological comparison of Schistosoma haematobium, Schistosoma bovis and Echinostoma caproni

PubMed Central

HIGÓN, MELISSA; COWAN, GRAEME; NAUSCH, NORMAN; CAVANAGH, DAVID; OLEAGA, ANA; TOLEDO, RAFAEL; STOTHARD, J. RUSSELL; ANTÚNEZ, ORETO; MARCILLA, ANTONIO; BURCHMORE, RICHARD; MUTAPI, FRANCISCA

2011-01-01

SUMMARY With the current paucity of vaccine targets for parasitic diseases, particularly those in childhood, the aim of this study was to compare protein expression and immune cross-reactivity between the trematodes Schistosoma haematobium, S. bovis and Echinostoma caproni in the hope of identifying novel intervention targets. Native adult parasite proteins were separated by 2-dimensional gel electrophoresis and identified through electrospray ionisation tandem mass spectrometry to produce a reference gel. Proteins from differential gel electrophoresis analyses of the three parasite proteomes were compared and screened against sera from hamsters infected with S. haematobium and E. caproni following 2-dimensional Western blotting. Differential protein expression between the three species was observed with circa 5% of proteins from S. haematobium showing expression up-regulation compared to the other two species. There was 91% similarity between the proteomes of the two Schistosoma species and 81% and 78·6% similarity between S. haematobium and S. bovis versus E. caproni, respectively. Although there were some common cross-species antigens, species-species targets were revealed which, despite evolutionary homology, could be due to phenotypic plasticity arising from different host-parasite relationships. Nevertheless, this approach helps to identify novel intervention targets which could be used as broad-spectrum candidates for future use in human and veterinary vaccines. PMID:21729355

PRIDE Inspector Toolsuite: Moving Toward a Universal Visualization Tool for Proteomics Data Standard Formats and Quality Assessment of ProteomeXchange Datasets.

PubMed

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.

PRIDE Inspector Toolsuite: Moving Toward a Universal Visualization Tool for Proteomics Data Standard Formats and Quality Assessment of ProteomeXchange Datasets*

PubMed Central

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/. PMID:26545397

Proteome-wide Subcellular Topologies of E. coli Polypeptides Database (STEPdb)*

PubMed Central

Orfanoudaki, Georgia; Economou, Anastassios

2014-01-01

Cell compartmentalization serves both the isolation and the specialization of cell functions. After synthesis in the cytoplasm, over a third of all proteins are targeted to other subcellular compartments. Knowing how proteins are distributed within the cell and how they interact is a prerequisite for understanding it as a whole. Surface and secreted proteins are important pathogenicity determinants. Here we present the STEP database (STEPdb) that contains a comprehensive characterization of subcellular localization and topology of the complete proteome of Escherichia coli. Two widely used E. coli proteomes (K-12 and BL21) are presented organized into thirteen subcellular classes. STEPdb exploits the wealth of genetic, proteomic, biochemical, and functional information on protein localization, secretion, and targeting in E. coli, one of the best understood model organisms. Subcellular annotations were derived from a combination of bioinformatics prediction, proteomic, biochemical, functional, topological data and extensive literature re-examination that were refined through manual curation. Strong experimental support for the location of 1553 out of 4303 proteins was based on 426 articles and some experimental indications for another 526. Annotations were provided for another 320 proteins based on firm bioinformatic predictions. STEPdb is the first database that contains an extensive set of peripheral IM proteins (PIM proteins) and includes their graphical visualization into complexes, cellular functions, and interactions. It also summarizes all currently known protein export machineries of E. coli K-12 and pairs them, where available, with the secretory proteins that use them. It catalogs the Sec- and TAT-utilizing secretomes and summarizes their topological features such as signal peptides and transmembrane regions, transmembrane topologies and orientations. It also catalogs physicochemical and structural features that influence topology such as abundance, solubility, disorder, heat resistance, and structural domain families. Finally, STEPdb incorporates prediction tools for topology (TMHMM, SignalP, and Phobius) and disorder (IUPred) and implements the BLAST2STEP that performs protein homology searches against the STEPdb. PMID:25210196

Beyond the Natural Proteome: Nondegenerate Saturation Mutagenesis-Methodologies and Advantages.

PubMed

Ferreira Amaral, M M; Frigotto, L; Hine, A V

2017-01-01

Beyond the natural proteome, high-throughput mutagenesis offers the protein engineer an opportunity to "tweak" the wild-type activity of a protein to create a recombinant protein with required attributes. Of the various approaches available, saturation mutagenesis is one of the core techniques employed by protein engineers, and in recent times, nondegenerate saturation mutagenesis is emerging as the approach of choice. This review compares the current methodologies available for conducting nondegenerate saturation mutagenesis with traditional, degenerate saturation and briefly outlines the options available for screening the resulting libraries, to discover a novel protein with the required activity and/or specificity. © 2017 Elsevier Inc. All rights reserved.

Analyses of Brucella Pathogenesis, Host Immunity, and Vaccine Targets using Systems Biology and Bioinformatics

PubMed Central

He, Yongqun

2011-01-01

Brucella is a Gram-negative, facultative intracellular bacterium that causes zoonotic brucellosis in humans and various animals. Out of 10 classified Brucella species, B. melitensis, B. abortus, B. suis, and B. canis are pathogenic to humans. In the past decade, the mechanisms of Brucella pathogenesis and host immunity have been extensively investigated using the cutting edge systems biology and bioinformatics approaches. This article provides a comprehensive review of the applications of Omics (including genomics, transcriptomics, and proteomics) and bioinformatics technologies for the analysis of Brucella pathogenesis, host immune responses, and vaccine targets. Based on more than 30 sequenced Brucella genomes, comparative genomics is able to identify gene variations among Brucella strains that help to explain host specificity and virulence differences among Brucella species. Diverse transcriptomics and proteomics gene expression studies have been conducted to analyze gene expression profiles of wild type Brucella strains and mutants under different laboratory conditions. High throughput Omics analyses of host responses to infections with virulent or attenuated Brucella strains have been focused on responses by mouse and cattle macrophages, bovine trophoblastic cells, mouse and boar splenocytes, and ram buffy coat. Differential serum responses in humans and rams to Brucella infections have been analyzed using high throughput serum antibody screening technology. The Vaxign reverse vaccinology has been used to predict many Brucella vaccine targets. More than 180 Brucella virulence factors and their gene interaction networks have been identified using advanced literature mining methods. The recent development of community-based Vaccine Ontology and Brucellosis Ontology provides an efficient way for Brucella data integration, exchange, and computer-assisted automated reasoning. PMID:22919594

Analyses of Brucella pathogenesis, host immunity, and vaccine targets using systems biology and bioinformatics.

PubMed

He, Yongqun

2012-01-01

Brucella is a Gram-negative, facultative intracellular bacterium that causes zoonotic brucellosis in humans and various animals. Out of 10 classified Brucella species, B. melitensis, B. abortus, B. suis, and B. canis are pathogenic to humans. In the past decade, the mechanisms of Brucella pathogenesis and host immunity have been extensively investigated using the cutting edge systems biology and bioinformatics approaches. This article provides a comprehensive review of the applications of Omics (including genomics, transcriptomics, and proteomics) and bioinformatics technologies for the analysis of Brucella pathogenesis, host immune responses, and vaccine targets. Based on more than 30 sequenced Brucella genomes, comparative genomics is able to identify gene variations among Brucella strains that help to explain host specificity and virulence differences among Brucella species. Diverse transcriptomics and proteomics gene expression studies have been conducted to analyze gene expression profiles of wild type Brucella strains and mutants under different laboratory conditions. High throughput Omics analyses of host responses to infections with virulent or attenuated Brucella strains have been focused on responses by mouse and cattle macrophages, bovine trophoblastic cells, mouse and boar splenocytes, and ram buffy coat. Differential serum responses in humans and rams to Brucella infections have been analyzed using high throughput serum antibody screening technology. The Vaxign reverse vaccinology has been used to predict many Brucella vaccine targets. More than 180 Brucella virulence factors and their gene interaction networks have been identified using advanced literature mining methods. The recent development of community-based Vaccine Ontology and Brucellosis Ontology provides an efficient way for Brucella data integration, exchange, and computer-assisted automated reasoning.

P2P proteomics -- data sharing for enhanced protein identification

PubMed Central

2012-01-01

Background In order to tackle the important and challenging problem in proteomics of identifying known and new protein sequences using high-throughput methods, we propose a data-sharing platform that uses fully distributed P2P technologies to share specifications of peer-interaction protocols and service components. By using such a platform, information to be searched is no longer centralised in a few repositories but gathered from experiments in peer proteomics laboratories, which can subsequently be searched by fellow researchers. Methods The system distributively runs a data-sharing protocol specified in the Lightweight Communication Calculus underlying the system through which researchers interact via message passing. For this, researchers interact with the system through particular components that link to database querying systems based on BLAST and/or OMSSA and GUI-based visualisation environments. We have tested the proposed platform with data drawn from preexisting MS/MS data reservoirs from the 2006 ABRF (Association of Biomolecular Resource Facilities) test sample, which was extensively tested during the ABRF Proteomics Standards Research Group 2006 worldwide survey. In particular we have taken the data available from a subset of proteomics laboratories of Spain's National Institute for Proteomics, ProteoRed, a network for the coordination, integration and development of the Spanish proteomics facilities. Results and Discussion We performed queries against nine databases including seven ProteoRed proteomics laboratories, the NCBI Swiss-Prot database and the local database of the CSIC/UAB Proteomics Laboratory. A detailed analysis of the results indicated the presence of a protein that was supported by other NCBI matches and highly scored matches in several proteomics labs. The analysis clearly indicated that the protein was a relatively high concentrated contaminant that could be present in the ABRF sample. This fact is evident from the information that could be derived from the proposed P2P proteomics system, however it is not straightforward to arrive to the same conclusion by conventional means as it is difficult to discard organic contamination of samples. The actual presence of this contaminant was only stated after the ABRF study of all the identifications reported by the laboratories. PMID:22293032

Direct Detection of Alternative Open Reading Frames Translation Products in Human Significantly Expands the Proteome

PubMed Central

Vanderperre, Benoît; Lucier, Jean-François; Bissonnette, Cyntia; Motard, Julie; Tremblay, Guillaume; Vanderperre, Solène; Wisztorski, Maxence; Salzet, Michel; Boisvert, François-Michel; Roucou, Xavier

2013-01-01

A fully mature mRNA is usually associated to a reference open reading frame encoding a single protein. Yet, mature mRNAs contain unconventional alternative open reading frames (AltORFs) located in untranslated regions (UTRs) or overlapping the reference ORFs (RefORFs) in non-canonical +2 and +3 reading frames. Although recent ribosome profiling and footprinting approaches have suggested the significant use of unconventional translation initiation sites in mammals, direct evidence of large-scale alternative protein expression at the proteome level is still lacking. To determine the contribution of alternative proteins to the human proteome, we generated a database of predicted human AltORFs revealing a new proteome mainly composed of small proteins with a median length of 57 amino acids, compared to 344 amino acids for the reference proteome. We experimentally detected a total of 1,259 alternative proteins by mass spectrometry analyses of human cell lines, tissues and fluids. In plasma and serum, alternative proteins represent up to 55% of the proteome and may be a potential unsuspected new source for biomarkers. We observed constitutive co-expression of RefORFs and AltORFs from endogenous genes and from transfected cDNAs, including tumor suppressor p53, and provide evidence that out-of-frame clones representing AltORFs are mistakenly rejected as false positive in cDNAs screening assays. Functional importance of alternative proteins is strongly supported by significant evolutionary conservation in vertebrates, invertebrates, and yeast. Our results imply that coding of multiple proteins in a single gene by the use of AltORFs may be a common feature in eukaryotes, and confirm that translation of unconventional ORFs generates an as yet unexplored proteome. PMID:23950983

The Human Skeletal Muscle Proteome Project: a reappraisal of the current literature

PubMed Central

Gonzalez‐Freire, Marta; Semba, Richard D.; Ubaida‐Mohien, Ceereena; Fabbri, Elisa; Scalzo, Paul; Højlund, Kurt; Dufresne, Craig; Lyashkov, Alexey

2016-01-01

Abstract Skeletal muscle is a large organ that accounts for up to half the total mass of the human body. A progressive decline in muscle mass and strength occurs with ageing and in some individuals configures the syndrome of ‘sarcopenia’, a condition that impairs mobility, challenges autonomy, and is a risk factor for mortality. The mechanisms leading to sarcopenia as well as myopathies are still little understood. The Human Skeletal Muscle Proteome Project was initiated with the aim to characterize muscle proteins and how they change with ageing and disease. We conducted an extensive review of the literature and analysed publically available protein databases. A systematic search of peer‐reviewed studies was performed using PubMed. Search terms included ‘human’, ‘skeletal muscle’, ‘proteome’, ‘proteomic(s)’, and ‘mass spectrometry’, ‘liquid chromatography‐mass spectrometry (LC‐MS/MS)’. A catalogue of 5431 non‐redundant muscle proteins identified by mass spectrometry‐based proteomics from 38 peer‐reviewed scientific publications from 2002 to November 2015 was created. We also developed a nosology system for the classification of muscle proteins based on localization and function. Such inventory of proteins should serve as a useful background reference for future research on changes in muscle proteome assessed by quantitative mass spectrometry‐based proteomic approaches that occur with ageing and diseases. This classification and compilation of the human skeletal muscle proteome can be used for the identification and quantification of proteins in skeletal muscle to discover new mechanisms for sarcopenia and specific muscle diseases that can be targeted for the prevention and treatment. PMID:27897395

Noninvasive biomarkers in non-alcoholic fatty liver disease: Current status and a glimpse of the future

PubMed Central

Fitzpatrick, Emer; Dhawan, Anil

2014-01-01

The development of non invasive biomarkers of disease has become a major focus of interest in nonalcoholic fatty liver disease (NAFLD). The large prevalence of the disease and the invasive nature of the investigation means that screening with liver biopsy is impractical. In addition to screening, the differentiation of those with simple steatosis vs steatohepatitis and fibrosis is clinically important as the prognosis of each differs. Serum biomarkers may be a combination of simple markers derived from large data sets or direct markers of disease activity. Serum markers of inflammation, apoptosis and oxidative stress in addition to fibrosis have been extensively studied in patients with NAFLD. Other techniques such as transient elastography, magnetic resonance elastography and acoustic radiation force imaging are becoming more established as noninvasive methods of detecting fibrosis in a variety of chronic liver conditions in addition to NAFLD. Newer high throughput methods such as proteomics and glycomics allow the nonhypothesis-driven identification of novel markers and may also potentially contribute to our understanding of the pathogenesis of the condition. This review addresses some of the methodological issues which need to be considered in the search for the ideal biomarker. It is likely that a combination of serum biomarkers and techniques such as transient elastography may provide the optimal diagnostic discrimination however this remains to be proven in large studies. PMID:25152587

Ecological distribution and population physiology defined by proteomics in a natural microbial community

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muller, R; Denef, Vincent; Kalnejals, Linda

An important challenge in microbial ecology is developing methods that simultaneously examine the physiology of organisms at the molecular level and their ecosystem level interactions in complex natural systems.We integrated extensive proteomic, geochemical, and biological information from 28 microbial communities collected from an acid mine drainage environment and representing a range of biofilm development stages and geochemical conditions to evaluate how the physiologies of the dominant and less abundant organisms change along environmental gradients. The initial colonist dominates across all environments, but its proteome changes between two stable states as communities diversify, implying that interspecies interactions affect this organism smore » metabolism. Its overall physiology is robust to abiotic environmental factors, but strong correlations exist between these factors and certain subsets of proteins, possibly accounting for its wide environmental distribution. Lower abundance populations are patchier in their distribution, and proteomic data indicate that their environmental niches may be constrained by specific sets of abiotic environmental factors. This research establishes an effective strategy to investigate ecological relationships between microbial physiology and the environment for whole communities in situ« less

Comparative Proteomic and Transcriptomic Analysis of Follistatin-Induced Skeletal Muscle Hypertrophy.

PubMed

Barbé, Caroline; Bray, Fabrice; Gueugneau, Marine; Devassine, Stéphanie; Lause, Pascale; Tokarski, Caroline; Rolando, Christian; Thissen, Jean-Paul

2017-10-06

Skeletal muscle, the most abundant body tissue, plays vital roles in locomotion and metabolism. Myostatin is a negative regulator of skeletal muscle mass. In addition to increasing muscle mass, Myostatin inhibition impacts muscle contractility and energy metabolism. To decipher the mechanisms of action of the Myostatin inhibitors, we used proteomic and transcriptomic approaches to investigate the changes induced in skeletal muscles of transgenic mice overexpressing Follistatin, a physiological Myostatin inhibitor. Our proteomic workflow included a fractionation step to identify weakly expressed proteins and a comparison of fast versus slow muscles. Functional annotation of altered proteins supports the phenotypic changes induced by Myostatin inhibition, including modifications in energy metabolism, fiber type, insulin and calcium signaling, as well as membrane repair and regeneration. Less than 10% of the differentially expressed proteins were found to be also regulated at the mRNA level but the Biological Process annotation, and the KEGG pathways analysis of transcriptomic results shows a great concordance with the proteomic data. Thus this study describes the most extensive omics analysis of muscle overexpressing Follistatin, providing molecular-level insights to explain the observed muscle phenotypic changes.

Ecological distribution and population physiology defined by proteomics in a natural microbial community

USGS Publications Warehouse

Mueller, Ryan S.; Denef, Vincent J.; Kalnejais, Linda H.; Suttle, K. Blake; Thomas, Brian C.; Wilmes, Paul; Smith, Richard L.; Nordstrom, D. Kirk; McCleskey, R. Blaine; Shah, Menesh B.; VerBekmoes, Nathan C.; Hettich, Robert L.; Banfield, Jillian F.

2010-01-01

An important challenge in microbial ecology is developing methods that simultaneously examine the physiology of organisms at the molecular level and their ecosystem level interactions in complex natural systems. We integrated extensive proteomic, geochemical, and biological information from 28 microbial communities collected from an acid mine drainage environment and representing a range of biofilm development stages and geochemical conditions to evaluate how the physiologies of the dominant and less abundant organisms change along environmental gradients. The initial colonist dominates across all environments, but its proteome changes between two stable states as communities diversify, implying that interspecies interactions affect this organism's metabolism. Its overall physiology is robust to abiotic environmental factors, but strong correlations exist between these factors and certain subsets of proteins, possibly accounting for its wide environmental distribution. Lower abundance populations are patchier in their distribution, and proteomic data indicate that their environmental niches may be constrained by specific sets of abiotic environmental factors. This research establishes an effective strategy to investigate ecological relationships between microbial physiology and the environment for whole communities in situ.

Comprehensive Proteomic Analysis of Human Milk-derived Extracellular Vesicles Unveils a Novel Functional Proteome Distinct from Other Milk Components*

PubMed Central

van Herwijnen, Martijn J.C.; Zonneveld, Marijke I.; Goerdayal, Soenita; Nolte – 't Hoen, Esther N.M.; Garssen, Johan; Stahl, Bernd; Maarten Altelaar, A.F.; Redegeld, Frank A.; Wauben, Marca H.M.

2016-01-01

Breast milk contains several macromolecular components with distinctive functions, whereby milk fat globules and casein micelles mainly provide nutrition to the newborn, and whey contains molecules that can stimulate the newborn's developing immune system and gastrointestinal tract. Although extracellular vesicles (EV) have been identified in breast milk, their physiological function and composition has not been addressed in detail. EV are submicron sized vehicles released by cells for intercellular communication via selectively incorporated lipids, nucleic acids, and proteins. Because of the difficulty in separating EV from other milk components, an in-depth analysis of the proteome of human milk-derived EV is lacking. In this study, an extensive LC-MS/MS proteomic analysis was performed of EV that had been purified from breast milk of seven individual donors using a recently established, optimized density-gradient-based EV isolation protocol. A total of 1963 proteins were identified in milk-derived EV, including EV-associated proteins like CD9, Annexin A5, and Flotillin-1, with a remarkable overlap between the different donors. Interestingly, 198 of the identified proteins are not present in the human EV database Vesiclepedia, indicating that milk-derived EV harbor proteins not yet identified in EV of different origin. Similarly, the proteome of milk-derived EV was compared with that of other milk components. For this, data from 38 published milk proteomic studies were combined in order to construct the total milk proteome, which consists of 2698 unique proteins. Remarkably, 633 proteins identified in milk-derived EV have not yet been identified in human milk to date. Interestingly, these novel proteins include proteins involved in regulation of cell growth and controlling inflammatory signaling pathways, suggesting that milk-derived EVs could support the newborn's developing gastrointestinal tract and immune system. Overall, this study provides an expansion of the whole milk proteome and illustrates that milk-derived EV are macromolecular components with a unique functional proteome. PMID:27601599

Extensive T-Cell Epitope Repertoire Sharing among Human Proteome, Gastrointestinal Microbiome, and Pathogenic Bacteria: Implications for the Definition of Self

PubMed Central

Bremel, Robert D.; Homan, E. Jane

2015-01-01

T-cell receptor binding to MHC-bound peptides plays a key role in discrimination between self and non-self. Only a subset, typically a pentamer, of amino acids in a MHC-bound peptide form the motif exposed to the T-cell receptor. We categorize and compare the T-cell exposed amino acid motif repertoire of the total proteomes of two groups of bacteria, comprising pathogens and gastrointestinal microbiome organisms, with the human proteome and immunoglobulins. Given the maximum 205, or 3.2 million of such motifs that bind T-cell receptors, there is considerable overlap in motif usage. We show that the human proteome, exclusive of immunoglobulins, only comprises three quarters of the possible motifs, of which 65.3% are also present in both composite bacterial proteomes. Very few motifs are unique to the human proteome. Immunoglobulin variable regions carry a broad diversity of T-cell exposed motifs (TCEMs) that provides a stratified random sample of the motifs found in pathogens, microbiome, and the human proteome. Individual bacterial genera and species vary in the content of immunoglobulin and human proteome matched motifs that they carry. Mycobacteria and Burkholderia spp carry a particularly high content of such matched motifs. Some bacteria retain a unique motif signature and motif sharing pattern with the human proteome. The implication is that distinguishing self from non-self does not depend on individual TCEMs, but on a complex and dynamic overlay of signals wherein the same TCEM may play different roles in different organisms, and the frequency with which a particular TCEM appears influences its effect. The patterns observed provide clues to bacterial immune evasion and to strategies for intervention, including vaccine design. The breadth and distinct frequency patterns of the immunoglobulin-derived peptides suggest a role of immunoglobulins in maintaining a broadly responsive T-cell repertoire. PMID:26557118

The Seminal fluid proteome of the polyandrous Red junglefowl offers insights into the molecular basis of fertility, reproductive ageing and domestication

PubMed Central

Borziak, Kirill; Álvarez-Fernández, Aitor; L. Karr, Timothy; Pizzari, Tommaso; Dorus, Steve

2016-01-01

Seminal fluid proteins (SFPs) are emerging as fundamental contributors to sexual selection given their role in post-mating reproductive events, particularly in polyandrous species where the ejaculates of different males compete for fertilisation. SFP identification however remains taxonomically limited and little is known about avian SFPs, despite extensive work on sexual selection in birds. We characterize the SF proteome of the polyandrous Red junglefowl, Gallus gallus, the wild species that gave rise to the domestic chicken. We identify 1,141 SFPs, including proteins involved in immunity and antimicrobial defences, sperm maturation, and fertilisation, revealing a functionally complex SF proteome. This includes a predominant contribution of blood plasma proteins that is conserved with human SF. By comparing the proteome of young and old males with fast or slow sperm velocity in a balanced design, we identify proteins associated with ageing and sperm velocity, and show that old males that retain high sperm velocity have distinct proteome characteristics. SFP comparisons with domestic chickens revealed both qualitative and quantitative differences likely associated with domestication and artificial selection. Collectively, these results shed light onto the functional complexity of avian SF, and provide a platform for molecular studies of fertility, reproductive ageing, and domestication. PMID:27804984

Growth phase-dependent proteomes of the Malaysian isolated Lactococcus lactis dairy strain M4 using label-free qualitative shotgun proteomics analysis.

PubMed

Yap, Theresa Wan Chen; Rabu, Amir; Abu Bakar, Farah Diba; Rahim, Raha Abdul; Mahadi, Nor Muhammad; Illias, Rosli Md; Murad, Abdul Munir Abdul

2014-01-01

Lactococcus lactis is the most studied mesophilic fermentative lactic acid bacterium. It is used extensively in the food industry and plays a pivotal role as a cell factory and also as vaccine delivery platforms. The proteome of the Malaysian isolated L. lactis M4 dairy strain, obtained from the milk of locally bred cows, was studied to elucidate the physiological changes occurring between the growth phases of this bacterium. In this study, ultraperformance liquid chromatography nanoflow electrospray ionization tandem mass spectrometry (UPLC- nano-ESI-MS(E)) approach was used for qualitative proteomic analysis. A total of 100 and 121 proteins were identified from the midexponential and early stationary growth phases, respectively, of the L. lactis strain M4. During the exponential phase, the most important reaction was the generation of sufficient energy, whereas, in the early stationary phase, the metabolic energy pathways decreased and the biosynthesis of proteins became more important. Thus, the metabolism of the cells shifted from energy production in the exponential phase to the synthesis of macromolecules in the stationary phase. The resultant proteomes are essential in providing an improved view of the cellular machinery of L. lactis during the transition of growth phases and hence provide insight into various biotechnological applications.

The Seminal fluid proteome of the polyandrous Red junglefowl offers insights into the molecular basis of fertility, reproductive ageing and domestication.

PubMed

Borziak, Kirill; Álvarez-Fernández, Aitor; L Karr, Timothy; Pizzari, Tommaso; Dorus, Steve

2016-11-02

Seminal fluid proteins (SFPs) are emerging as fundamental contributors to sexual selection given their role in post-mating reproductive events, particularly in polyandrous species where the ejaculates of different males compete for fertilisation. SFP identification however remains taxonomically limited and little is known about avian SFPs, despite extensive work on sexual selection in birds. We characterize the SF proteome of the polyandrous Red junglefowl, Gallus gallus, the wild species that gave rise to the domestic chicken. We identify 1,141 SFPs, including proteins involved in immunity and antimicrobial defences, sperm maturation, and fertilisation, revealing a functionally complex SF proteome. This includes a predominant contribution of blood plasma proteins that is conserved with human SF. By comparing the proteome of young and old males with fast or slow sperm velocity in a balanced design, we identify proteins associated with ageing and sperm velocity, and show that old males that retain high sperm velocity have distinct proteome characteristics. SFP comparisons with domestic chickens revealed both qualitative and quantitative differences likely associated with domestication and artificial selection. Collectively, these results shed light onto the functional complexity of avian SF, and provide a platform for molecular studies of fertility, reproductive ageing, and domestication.

Proteomic and metallomic strategies for understanding the mode of action of anticancer metallodrugs.

PubMed

Gabbiani, Chiara; Magherini, Francesca; Modesti, Alessandra; Messori, Luigi

2010-05-01

Since the discovery of cisplatin and its introduction in the clinics, metal compounds have been intensely investigated in view of their possible application in cancer therapy. In this frame, a deeper understanding of their mode of action, still rather obscure, might turn crucial for the design and the obtainment of new and better anticancer agents. Due to the extreme complexity of the biological systems, it is now widely accepted that innovative and information-rich methods are absolutely needed to afford such a goal. Recently, both proteomic and metallomic strategies were successfully implemented for the elucidation of specific mechanistic features of anticancer metallodrugs within an innovative "Systems Biology" perspective. Particular attention was paid to the following issues: i) proteomic studies of the molecular basis of platinum resistance; ii) proteomic analysis of cellular responses to cytotoxic metallodrugs; iii) metallomic studies of the transformation and fate of metallodrugs in cellular systems. Notably, those pioneering studies, that are reviewed here, allowed a significant progress in the understanding of the molecular mechanisms of metal based drugs at the cellular level. A further extension of those studies and a closer integration of proteomic and metallomic strategies and technologies might realistically lead to rapid and significant advancements in the mechanistic knowledge of anticancer metallodrugs.

Suberoylanilide hydroxamic acid treatment reveals crosstalks among proteome, ubiquitylome and acetylome in non-small cell lung cancer A549 cell line.

PubMed

Wu, Quan; Cheng, Zhongyi; Zhu, Jun; Xu, Weiqing; Peng, Xiaojun; Chen, Chuangbin; Li, Wenting; Wang, Fengsong; Cao, Lejie; Yi, Xingling; Wu, Zhiwei; Li, Jing; Fan, Pingsheng

2015-03-31

Suberoylanilide hydroxamic acid (SAHA) is a well-known histone deacetylase (HDAC) inhibitor and has been used as practical therapy for breast cancer and non-small cell lung cancer (NSCLC). It is previously demonstrated that SAHA treatment could extensively change the profile of acetylome and proteome in cancer cells. However, little is known about the impact of SAHA on other protein modifications and the crosstalks among different modifications and proteome, hindering the deep understanding of SAHA-mediated cancer therapy. In this work, by using SILAC technique, antibody-based affinity enrichment and high-resolution LC-MS/MS analysis, we investigated quantitative proteome, acetylome and ubiquitylome as well as crosstalks among the three datasets in A549 cells toward SAHA treatment. In total, 2968 proteins, 1099 acetylation sites and 1012 ubiquitination sites were quantified in response to SAHA treatment, respectively. With the aid of intensive bioinformatics, we revealed that the proteome and ubiquitylome were negatively related upon SAHA treatment. Moreover, the impact of SAHA on acetylome resulted in 258 up-regulated and 99 down-regulated acetylation sites at the threshold of 1.5 folds. Finally, we identified 55 common sites with both acetylation and ubiquitination, among which ubiquitination level in 43 sites (78.2%) was positive related to acetylation level.

Global Analysis of Salmonella Alternative Sigma Factor E on Protein Translation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Jie; Nakayasu, Ernesto S.; Overall, Christopher C.

The alternative sigma factor E (σ E) is critical for response to extracytoplasmic stress in Salmonella. Extensive studies have been conducted on σ E-regulated gene expression, particularly at the transcriptional level. Increasing evidence suggests however that σ E may indirectly participate in post-transcriptional regulation. Here in this study, we conducted sample-matched global proteomic and transcriptomic analyses to determine the level of regulation mediated by σ E in Salmonella. We analysed samples from wild type and isogenic rpoE mutant Salmonella cultivated in three different conditions; nutrient-rich and conditions that mimic early and late intracellular infection. We found that 30% of themore » observed proteome was regulated by σ E combining all three conditions. In different growth conditions, σ E affected the expression of a broad spectrum of Salmonella proteins required for miscellaneous functions. Those involved in transport and binding, protein synthesis, and stress response were particularly highlighted. By comparing transcriptomic and proteomic data, we identified genes post-transcriptionally regulated by σ E and found that post-transcriptional regulation was responsible for a majority of changes observed in the σ E-regulated proteome. Further, comparison of transcriptomic and proteomic data from hfq mutant of Salmonella demonstrated that σ E–mediated post-transcriptional regulation was partially dependent on the RNA-binding protein Hfq.« less

Global Analysis of Salmonella Alternative Sigma Factor E on Protein Translation

DOE PAGES

Li, Jie; Nakayasu, Ernesto S.; Overall, Christopher C.; ...

2015-02-16

The alternative sigma factor E (σ E) is critical for response to extracytoplasmic stress in Salmonella. Extensive studies have been conducted on σ E-regulated gene expression, particularly at the transcriptional level. Increasing evidence suggests however that σ E may indirectly participate in post-transcriptional regulation. Here in this study, we conducted sample-matched global proteomic and transcriptomic analyses to determine the level of regulation mediated by σ E in Salmonella. We analysed samples from wild type and isogenic rpoE mutant Salmonella cultivated in three different conditions; nutrient-rich and conditions that mimic early and late intracellular infection. We found that 30% of themore » observed proteome was regulated by σ E combining all three conditions. In different growth conditions, σ E affected the expression of a broad spectrum of Salmonella proteins required for miscellaneous functions. Those involved in transport and binding, protein synthesis, and stress response were particularly highlighted. By comparing transcriptomic and proteomic data, we identified genes post-transcriptionally regulated by σ E and found that post-transcriptional regulation was responsible for a majority of changes observed in the σ E-regulated proteome. Further, comparison of transcriptomic and proteomic data from hfq mutant of Salmonella demonstrated that σ E–mediated post-transcriptional regulation was partially dependent on the RNA-binding protein Hfq.« less

Proteome-wide identification of predominant subcellular protein localizations in a bacterial model organism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stekhoven, Daniel J.; Omasits, Ulrich; Quebatte, Maxime

2014-03-01

Proteomics data provide unique insights into biological systems, including the predominant subcellular localization (SCL) of proteins, which can reveal important clues about their functions. Here we analyzed data of a complete prokaryotic proteome expressed under two conditions mimicking interaction of the emerging pathogen Bartonella henselae with its mammalian host. Normalized spectral count data from cytoplasmic, total membrane, inner and outer membrane fractions allowed us to identify the predominant SCL for 82% of the identified proteins. The spectral count proportion of total membrane versus cytoplasmic fractions indicated the propensity of cytoplasmic proteins to co-fractionate with the inner membrane, and enabled usmore » to distinguish cytoplasmic, peripheral innermembrane and bona fide inner membrane proteins. Principal component analysis and k-nearest neighbor classification training on selected marker proteins or predominantly localized proteins, allowed us to determine an extensive catalog of at least 74 expressed outer membrane proteins, and to extend the SCL assignment to 94% of the identified proteins, including 18% where in silico methods gave no prediction. Suitable experimental proteomics data combined with straightforward computational approaches can thus identify the predominant SCL on a proteome-wide scale. Finally, we present a conceptual approach to identify proteins potentially changing their SCL in a condition-dependent fashion.« less

Potential for Metabolomics-Based Markers of Exposure:Encouraging Evidence from Studies using Model Organisms

EPA Science Inventory

Genomic techniques (transcriptomics, proteomics, and metabolomics) have the potential to significantly improve the way chemical risk is managed in the 21st century. Indeed, a significant amount of research has been devoted to the use of these techniques to screen chemicals for h...

ITRAQ MASS SPECTROMETRIC PROTEOMIC APPLICATIONS FOR IN VIVO TOXICOLOGY STUDIES OF AMPHIBIAN SPECIES: DATA HANDLING AND INTERPRETATION USING PEPTIDE-TAGGING SOFTWARE

EPA Science Inventory

This addresses the USEPA's need for a cost effective, non-mammalian screening assay for thyroid axis disrupting chemicals; a multi-endpoint strategy combining molecular and in vivo protocols in an amphibian model is being applied at MED Duluth.

Genetic and Proteomic Interrogation of Lower Confidence Candidate Genes Reveals Signaling Networks in beta-Catenin-Active Cancers | Office of Cancer Genomics

Cancer.gov

Genome-scale expression studies and comprehensive loss-of-function genetic screens have focused almost exclusively on the highest confidence candidate genes. Here, we describe a strategy for characterizing the lower confidence candidates identified by such approaches.

Optimizing screening for tuberculosis and hepatitis B prior to starting tumor necrosis factor-α inhibitors in Crohn's disease.

PubMed

van der Have, Mike; Oldenburg, Bas; Fidder, Herma H; Belderbos, Tim D G; Siersema, Peter D; van Oijen, Martijn G H

2014-03-01

Treatment with tumor necrosis factor-α (TNF-α) inhibitors in patients with Crohn's disease (CD) is associated with potentially serious infections, including tuberculosis (TB) and hepatitis B virus (HBV). We assessed the cost-effectiveness of extensive TB screening and HBV screening prior to initiating TNF-α inhibitors in CD. We constructed two Markov models: (1) comparing tuberculin skin test (TST) combined with chest X-ray (conventional TB screening) versus TST and chest X-ray followed by the interferon-gamma release assay (extensive TB screening) in diagnosing TB; and (2) HBV screening versus no HBV screening. Our base-case included an adult CD patient starting with infliximab treatment. Input parameters were extracted from the literature. Direct medical costs were assessed and discounted following a third-party payer perspective. The main outcome was the incremental cost-effectiveness ratio (ICER). Sensitivity and Monte Carlo analyses were performed over wide ranges of probability and cost estimates. At base-case, the ICERs of extensive screening and HBV screening were €64,340 and €75,760 respectively to gain one quality-adjusted life year. Sensitivity analyses concluded that extensive TB screening was a cost-effective strategy if the latent TB prevalence is more than 12 % or if the false positivity rate of TST is more than 20 %. HBV screening became cost-effective if HBV reactivation or HBV-related mortality is higher than 37 and 62 %, respectively. Extensive TB screening and HBV screening are not cost-effective compared with conventional TB screening and no HBV screening, respectively. However, when targeted at high-risk patient groups, these screening strategies are likely to become cost-effective.

Combined analysis of transcriptome and proteome data as a tool for the identification of candidate biomarkers in renal cell carcinoma

PubMed Central

Seliger, Barbara; Dressler, Sven P.; Wang, Ena; Kellner, Roland; Recktenwald, Christian V.; Lottspeich, Friedrich; Marincola, Francesco M.; Baumgärtner, Maja; Atkins, Derek; Lichtenfels, Rudolf

2012-01-01

Results obtained from expression profilings of renal cell carcinoma using different “ome”-based approaches and comprehensive data analysis demonstrated that proteome-based technologies and cDNA microarray analyses complement each other during the discovery phase for disease-related candidate biomarkers. The integration of the respective data revealed the uniqueness and complementarities of the different technologies. While comparative cDNA microarray analyses though restricted to upregulated targets largely revealed genes involved in controlling gene/protein expression (19%) and signal transduction processes (13%), proteomics/PROTEOMEX-defined candidate biomarkers include enzymes of the cellular metabolism (36%), transport proteins (12%) and cell motility/structural molecules (10%). Candidate biomarkers defined by proteomics and PROTEOMEX are frequently shared, whereas the sharing rate between cDNA microarray and proteome-based profilings is limited. Putative candidate biomarkers provide insights into their cellular (dys)function and their diagnostic/prognostic value but still warrant further validation in larger patient numbers. Based on the fact that merely 3 candidate biomarkers were shared by all applied technologies, namely annexin A4, tubulin alpha-1A chain and ubiquitin carboxyl-terminal hydrolase L1 the analysis at a single hierarchical level of biological regulation seems to provide only limited results thus emphasizing the importance and benefit of performing rather combinatorial screenings which can complement the standard clinical predictors. PMID:19235166

Proteomics for exploiting diversity of lupin seed storage proteins and their use as nutraceuticals for health and welfare.

PubMed

Cabello-Hurtado, Francisco; Keller, Jean; Ley, José; Sanchez-Lucas, Rosa; Jorrín-Novo, Jesús V; Aïnouche, Abdelkader

2016-06-30

Lupins have a variety of both traditional and modern uses. In the last decade, reports assessing the benefits of lupin seed proteins have proliferated and, nowadays, the pharmaceutical industry is interested in lupin proteins for human health. Modern genomics and proteomics have hugely contributed to describing the diversity of lupin storage genes and, above all, proteins. Most of these studies have been centered on few edible lupin species. However, Lupinus genus comprises hundreds of species spread throughout the Old and New Worlds, and these resources have been scarcely explored and exploited. We present here a detailed review of the literature on the potential of lupin seed proteins as nutraceuticals, and the use of -omic tools to analyze seed storage polypeptides in main edible lupins and their diversity at the Lupinus inter- and intra-species level. In this sense, proteomics, more than any other, has been a key approach. Proteomics has shown that lupin seed protein diversity, where post-translational modifications yield a large number of peptide variants with a potential concern in bioactivity, goes far beyond gene diversity. The future extended use of second and third generation proteomics should definitely help to go deeper into coverage and characterization of lupin seed proteome. Some important topics concerning storage proteins from lupin seeds are presented and analyzed in an integrated way in this review. Proteomic approaches have been essential in characterizing lupin seed protein diversity, which goes far beyond gene diversity since the protein level adds to the latter differential proteolytic cleavage of conglutin pro-proteins and a diverse array of glycosylation forms and sites. Proteomics has also proved helpful for screening and studying Lupinus germplasm with the future aim of exploiting and improving food production, quality, and nutritional values. Copyright © 2016 Elsevier B.V. All rights reserved.

A Bayesian algorithm for detecting differentially expressed proteins and its application in breast cancer research

NASA Astrophysics Data System (ADS)

Santra, Tapesh; Delatola, Eleni Ioanna

2016-07-01

Presence of considerable noise and missing data points make analysis of mass-spectrometry (MS) based proteomic data a challenging task. The missing values in MS data are caused by the inability of MS machines to reliably detect proteins whose abundances fall below the detection limit. We developed a Bayesian algorithm that exploits this knowledge and uses missing data points as a complementary source of information to the observed protein intensities in order to find differentially expressed proteins by analysing MS based proteomic data. We compared its accuracy with many other methods using several simulated datasets. It consistently outperformed other methods. We then used it to analyse proteomic screens of a breast cancer (BC) patient cohort. It revealed large differences between the proteomic landscapes of triple negative and Luminal A, which are the most and least aggressive types of BC. Unexpectedly, majority of these differences could be attributed to the direct transcriptional activity of only seven transcription factors some of which are known to be inactive in triple negative BC. We also identified two new proteins which significantly correlated with the survival of BC patients, and therefore may have potential diagnostic/prognostic values.

Salivary biomarker development using genomic, proteomic and metabolomic approaches

PubMed Central

2012-01-01

The use of saliva as a diagnostic sample provides a non-invasive, cost-efficient method of sample collection for disease screening without the need for highly trained professionals. Saliva collection is far more practical and safe compared with invasive methods of sample collection, because of the infection risk from contaminated needles during, for example, blood sampling. Furthermore, the use of saliva could increase the availability of accurate diagnostics for remote and impoverished regions. However, the development of salivary diagnostics has required technical innovation to allow stabilization and detection of analytes in the complex molecular mixture that is saliva. The recent development of cost-effective room temperature analyte stabilization methods, nucleic acid pre-amplification techniques and direct saliva transcriptomic analysis have allowed accurate detection and quantification of transcripts found in saliva. Novel protein stabilization methods have also facilitated improved proteomic analyses. Although candidate biomarkers have been discovered using epigenetic, transcriptomic, proteomic and metabolomic approaches, transcriptomic analyses have so far achieved the most progress in terms of sensitivity and specificity, and progress towards clinical implementation. Here, we review recent developments in salivary diagnostics that have been accomplished using genomic, transcriptomic, proteomic and metabolomic approaches. PMID:23114182

Proteome screening of pleural effusions identifies IL1A as a diagnostic biomarker for non-small cell lung cancer.

PubMed

Li, Yuanyuan; Lian, Hengning; Jia, Qingzhu; Wan, Ying

2015-02-06

Non-small cell lung cancer (NSCLC) is a common malignant disease, and in ~10-20% of patients, pleural effusion is the first symptom. The pleural effusion proteome contains information on pulmonary disease that directly or indirectly reflects pathophysiological status. However, the proteome of pleural effusion in NSCLC patients is not well understood, nor is the variability in protein composition between malignant and benign pleural effusions. Here, we investigated the different proteins in pleural effusions from NSCLC and tuberculosis (TB) patients by using nano-scale liquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis. In total, 363 proteins were identified in the NSCLC pleural effusion proteome with a low false discovery rate (<1%), and 199 proteins were unique to NSCLC. The proteins in the NSCLC patients' pleural effusion were involved in cell adhesion, proteolysis, and cell migration. Furthermore, interleukin 1 alpha (IL1A), a protein that regulates tumor growth, angiogenesis, and metastasis, was significantly more abundant in the NSCLC group compared to the TB group, a finding that was validated with an ELISA assay. Copyright © 2014 Elsevier Inc. All rights reserved.

Amyloidosis: Insights from Proteomics.

PubMed

Dogan, Ahmet

2017-01-24

Amyloidoses are a spectrum of disorders caused by abnormal folding and extracellular deposition of proteins. The deposits lead to tissue damage and organ dysfunction, particularly in the heart, kidneys, and nerves. There are at least 30 different proteins that can cause amyloidosis. The clinical management depends entirely on the type of protein deposited, and thus on the underlying pathogenesis, and often requires high-risk therapeutic intervention. Application of mass spectrometry-based proteomic technologies for analysis of amyloid plaques has transformed the way amyloidosis is diagnosed and classified. Proteomic assays have been extensively used for clinical management of patients with amyloidosis, providing unprecedented diagnostic and biological information. They have shed light on the pathogenesis of different amyloid types and have led to identification of numerous new amyloid types, including ALECT2 amyloidosis, which is now recognized as one of the most common causes of systemic amyloidosis in North America.

Identification and characterization of potential druggable targets among hypothetical proteins of extensively drug resistant Mycobacterium tuberculosis (XDR KZN 605) through subtractive genomics approach.

PubMed

Uddin, Reaz; Siddiqui, Quratulain Nehal; Azam, Syed Sikander; Saima, Bibi; Wadood, Abdul

2018-03-01

Among the resistant isolates of tuberculosis (TB), the multidrug resistance tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB) are the areas of growing concern for which the front-line antibiotics are no more effective. As a result, the search of new therapeutic targets against TB is an imperative need of time. On the other hand, the target identification is an a priori step in drug discovery based research. Furthermore, the availability of the complete proteomic data of extensively drug resistant Mycobacterium tuberculosis (XDR-MTB) made it possible to carry out in silico analysis for the discovery of new drug targets. In the current study, we aimed to prioritize the potential drug targets among the hypothetical proteins of XDR-TB via subtractive genomics approach. In the subtractive genomics, we stepwise reduced the complete proteome of XDR-MTB to only two hypothetical proteins and evidently proposed them as new therapeutic targets. The 3D structure of one of the two target proteins was predicted via homology modeling and later on, validated by various analysis tools. Our study suggested that the domains identified and the motif hits found in the sequences of the shortlisted drug targets are crucial for the survival of the XDR-MTB. To the best of our knowledge, the current study is the first attempt in which the complete proteomic data of XDR-MTB was subjected to the computational subtractive genomics approach and therefore, would provide an opportunity to identify the unique therapeutic targets against deadly XDR-MTB. Copyright © 2017 Elsevier B.V. All rights reserved.

Screening of entomopathogenic Metarhizium anisopliae isolates and proteomic analysis of secretion synthesized in response to cowpea weevil (Callosobruchus maculatus) exoskeleton.

PubMed

Murad, André M; Laumann, Raul A; Lima, Thaina de A; Sarmento, Rubia B C; Noronha, Eliane F; Rocha, Thales L; Valadares-Inglis, Maria C; Franco, Octávio L

2006-01-01

Cowpea crops are severely attacked by Callosobruchus maculatus, a Coleopteran that at the larval stage penetrates into stored seeds and feeds on cotyledons. Cowpea weevil control could be based in utilization of bacteria and fungi to reduce pest development. Entomopathogenic fungi, such as Metarhizium anisopliae, are able to control insect-pests and are widely applied in biological control. This report evaluated ten M. anisopliae isolates according to their virulence, correlating chitinolytic, proteolytic and alpha-amylolytic activities, as well proteomic analysis by two dimensional gels of fungal secretions in response to an induced medium containing C. maculatus shells, indicating novel biotechnological tools capable of improving cowpea crop resistance.

Equine recurrent uveitis--a spontaneous horse model of uveitis.

PubMed

Deeg, Cornelia A; Hauck, Stefanie M; Amann, Barbara; Pompetzki, Dirk; Altmann, Frank; Raith, Albert; Schmalzl, Thomas; Stangassinger, Manfred; Ueffing, Marius

2008-01-01

Equine recurrent uveitis (ERU) is an autoimmune disease that occurs with a high prevalence (10%) in horses. ERU represents the only reliable spontaneous model for human autoimmune uveitis. We already identified and characterized novel autoantigens (malate dehydrogenase, recoverin, CRALBP) by analyzing the autoantibody-binding pattern of horses affected by spontaneous recurrent uveitis (ERU) to the retinal proteome. CRALBP also seems to be relevant to human autoimmune uveitis. Proteomic screening of vitreous and retinal samples from ERU diseased cases in comparison to healthy controls has led to the identification of a series of differentially regulated proteins, which are functionally linked to the immune system and the maintenance of the blood-retinal barrier. 2008 S. Karger AG, Basel.

Microfluidic liquid chromatography system for proteomic applications and biomarker screening.

PubMed

Lazar, Iulia M; Trisiripisal, Phichet; Sarvaiya, Hetal A

2006-08-01

A microfluidic liquid chromatography (LC) system for proteomic investigations that integrates all the necessary components for stand-alone operation, i.e., pump, valve, separation column, and electrospray interface, is described in this paper. The overall size of the LC device is small enough to enable the integration of two fully functional separation systems on a 3 in. x 1 in. glass microchip. A multichannel architecture that uses electroosmotic pumping principles provides the necessary functionality for eluent propulsion and sample valving. The flow rates generated within these chips are fully consistent with the requirements of nano-LC platforms that are routinely used in proteomic applications. The microfluidic device was evaluated for the analysis of a protein digest obtained from the MCF7 breast cancer cell line. The cytosolic protein extract was processed according to a shotgun protocol, and after tryptic digestion and prefractionation using strong cation exchange chromatography (SCX), selected sample subfractions were analyzed with conventional and microfluidic LC platforms. Using similar experimental conditions, the performance of the microchip LC was comparable to that obtained with benchtop instrumentation, providing an overlap of 75% in proteins that were identified by more than two unique peptides. The microfluidic LC analysis of a protein-rich SCX fraction enabled the confident identification of 77 proteins by using conventional data filtering parameters, of 39 proteins with p < 0.001, and of 5 proteins that are known to be cancer-specific biomarkers, demonstrating thus the potential applicability of these chips for future high-throughput biomarker screening applications.

The mzTab data exchange format: communicating mass-spectrometry-based proteomics and metabolomics experimental results to a wider audience.

PubMed

Griss, Johannes; Jones, Andrew R; Sachsenberg, Timo; Walzer, Mathias; Gatto, Laurent; Hartler, Jürgen; Thallinger, Gerhard G; Salek, Reza M; Steinbeck, Christoph; Neuhauser, Nadin; Cox, Jürgen; Neumann, Steffen; Fan, Jun; Reisinger, Florian; Xu, Qing-Wei; Del Toro, Noemi; Pérez-Riverol, Yasset; Ghali, Fawaz; Bandeira, Nuno; Xenarios, Ioannis; Kohlbacher, Oliver; Vizcaíno, Juan Antonio; Hermjakob, Henning

2014-10-01

The HUPO Proteomics Standards Initiative has developed several standardized data formats to facilitate data sharing in mass spectrometry (MS)-based proteomics. These allow researchers to report their complete results in a unified way. However, at present, there is no format to describe the final qualitative and quantitative results for proteomics and metabolomics experiments in a simple tabular format. Many downstream analysis use cases are only concerned with the final results of an experiment and require an easily accessible format, compatible with tools such as Microsoft Excel or R. We developed the mzTab file format for MS-based proteomics and metabolomics results to meet this need. mzTab is intended as a lightweight supplement to the existing standard XML-based file formats (mzML, mzIdentML, mzQuantML), providing a comprehensive summary, similar in concept to the supplemental material of a scientific publication. mzTab files can contain protein, peptide, and small molecule identifications together with experimental metadata and basic quantitative information. The format is not intended to store the complete experimental evidence but provides mechanisms to report results at different levels of detail. These range from a simple summary of the final results to a representation of the results including the experimental design. This format is ideally suited to make MS-based proteomics and metabolomics results available to a wider biological community outside the field of MS. Several software tools for proteomics and metabolomics have already adapted the format as an output format. The comprehensive mzTab specification document and extensive additional documentation can be found online. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Complex and extensive post-transcriptional regulation revealed by integrative proteomic and transcriptomic analysis of metabolite stress response in Clostridium acetobutylicum.

PubMed

Venkataramanan, Keerthi P; Min, Lie; Hou, Shuyu; Jones, Shawn W; Ralston, Matthew T; Lee, Kelvin H; Papoutsakis, E Terry

2015-01-01

Clostridium acetobutylicum is a model organism for both clostridial biology and solvent production. The organism is exposed to its own toxic metabolites butyrate and butanol, which trigger an adaptive stress response. Integrative analysis of proteomic and RNAseq data may provide novel insights into post-transcriptional regulation. The identified iTRAQ-based quantitative stress proteome is made up of 616 proteins with a 15 % genome coverage. The differentially expressed proteome correlated poorly with the corresponding differential RNAseq transcriptome. Up to 31 % of the differentially expressed proteins under stress displayed patterns opposite to those of the transcriptome, thus suggesting significant post-transcriptional regulation. The differential proteome of the translation machinery suggests that cells employ a different subset of ribosomal proteins under stress. Several highly upregulated proteins but with low mRNA levels possessed mRNAs with long 5'UTRs and strong RBS scores, thus supporting the argument that regulatory elements on the long 5'UTRs control their translation. For example, the oxidative stress response rubrerythrin was upregulated only at the protein level up to 40-fold without significant mRNA changes. We also identified many leaderless transcripts, several displaying different transcriptional start sites, thus suggesting mRNA-trimming mechanisms under stress. Downregulation of Rho and partner proteins pointed to changes in transcriptional elongation and termination under stress. The integrative proteomic-transcriptomic analysis demonstrated complex expression patterns of a large fraction of the proteome. Such patterns could not have been detected with one or the other omic analyses. Our analysis proposes the involvement of specific molecular mechanisms of post-transcriptional regulation to explain the observed complex stress response.

The mzTab Data Exchange Format: Communicating Mass-spectrometry-based Proteomics and Metabolomics Experimental Results to a Wider Audience*

PubMed Central

Griss, Johannes; Jones, Andrew R.; Sachsenberg, Timo; Walzer, Mathias; Gatto, Laurent; Hartler, Jürgen; Thallinger, Gerhard G.; Salek, Reza M.; Steinbeck, Christoph; Neuhauser, Nadin; Cox, Jürgen; Neumann, Steffen; Fan, Jun; Reisinger, Florian; Xu, Qing-Wei; del Toro, Noemi; Pérez-Riverol, Yasset; Ghali, Fawaz; Bandeira, Nuno; Xenarios, Ioannis; Kohlbacher, Oliver; Vizcaíno, Juan Antonio; Hermjakob, Henning

2014-01-01

The HUPO Proteomics Standards Initiative has developed several standardized data formats to facilitate data sharing in mass spectrometry (MS)-based proteomics. These allow researchers to report their complete results in a unified way. However, at present, there is no format to describe the final qualitative and quantitative results for proteomics and metabolomics experiments in a simple tabular format. Many downstream analysis use cases are only concerned with the final results of an experiment and require an easily accessible format, compatible with tools such as Microsoft Excel or R. We developed the mzTab file format for MS-based proteomics and metabolomics results to meet this need. mzTab is intended as a lightweight supplement to the existing standard XML-based file formats (mzML, mzIdentML, mzQuantML), providing a comprehensive summary, similar in concept to the supplemental material of a scientific publication. mzTab files can contain protein, peptide, and small molecule identifications together with experimental metadata and basic quantitative information. The format is not intended to store the complete experimental evidence but provides mechanisms to report results at different levels of detail. These range from a simple summary of the final results to a representation of the results including the experimental design. This format is ideally suited to make MS-based proteomics and metabolomics results available to a wider biological community outside the field of MS. Several software tools for proteomics and metabolomics have already adapted the format as an output format. The comprehensive mzTab specification document and extensive additional documentation can be found online. PMID:24980485

Global Reprogramming of Host SUMOylation during Influenza Virus Infection

PubMed Central

Domingues, Patricia; Golebiowski, Filip; Tatham, Michael H.; Lopes, Antonio M.; Taggart, Aislynn; Hay, Ronald T.; Hale, Benjamin G.

2015-01-01

Summary Dynamic nuclear SUMO modifications play essential roles in orchestrating cellular responses to proteotoxic stress, DNA damage, and DNA virus infection. Here, we describe a non-canonical host SUMOylation response to the nuclear-replicating RNA pathogen, influenza virus, and identify viral RNA polymerase activity as a major contributor to SUMO proteome remodeling. Using quantitative proteomics to compare stress-induced SUMOylation responses, we reveal that influenza virus infection triggers unique re-targeting of SUMO to 63 host proteins involved in transcription, mRNA processing, RNA quality control, and DNA damage repair. This is paralleled by widespread host deSUMOylation. Depletion screening identified ten virus-induced SUMO targets as potential antiviral factors, including C18orf25 and the SMC5/6 and PAF1 complexes. Mechanistic studies further uncovered a role for SUMOylation of the PAF1 complex component, parafibromin (CDC73), in potentiating antiviral gene expression. Our global characterization of influenza virus-triggered SUMO redistribution provides a proteomic resource to understand host nuclear SUMOylation responses to infection. PMID:26549460

Proteomic evaluation of genetically modified crops: current status and challenges

PubMed Central

Gong, Chun Yan; Wang, Tai

2013-01-01

Hectares of genetically modified (GM) crops have increased exponentially since 1996, when such crops began to be commercialized. GM biotechnology, together with conventional breeding, has become the main approach to improving agronomic traits of crops. However, people are concerned about the safety of GM crops, especially GM-derived food and feed. Many efforts have been made to evaluate the unintended effects caused by the introduction of exogenous genes. “Omics” techniques have advantages over targeted analysis in evaluating such crops because of their use of high-throughput screening. Proteins are key players in gene function and are directly involved in metabolism and cellular development or have roles as toxins, antinutrients, or allergens, which are essential for human health. Thus, proteomics can be expected to become one of the most useful tools in safety assessment. This review assesses the potential of proteomics in evaluating various GM crops. We further describe the challenges in ensuring homogeneity and sensitivity in detection techniques. PMID:23471542

Proteomic profiling of human pleural effusion using two-dimensional nano liquid chromatography tandem mass spectrometry.

PubMed

Tyan, Yu-Chang; Wu, Hsin-Yi; Lai, Wu-Wei; Su, Wu-Chou; Liao, Pao-Chi

2005-01-01

Pleural effusion, an accumulation of pleural fluid, contains proteins originated from plasma filtrate and, especially when tissues are damaged, parenchyma interstitial spaces of lungs and/or other organs. This study details protein profiles in human pleural effusion from 43 lung adenocarcinoma patients by a two-dimensional nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry (2D nano-HPLC-ESI-MS/MS) system. The experimental results revealed the identification of 1415 unique proteins from human pleural effusion. Among these 124 proteins identified with higher confidence levels, some proteins have not been reported in plasma and may represent proteins specifically present in pleural effusion. These proteins are valuable for mass identification of differentially expressed proteins involved in proteomics database and screening biomarker to further study in human lung adenocarcinoma. The significance of the use of proteomics analysis of human pleural fluid for the search of new lung cancer marker proteins, and for their simultaneous display and analysis in patients suffering from lung disorders has been examined.

Proteomic evaluation of genetically modified crops: current status and challenges.

PubMed

Gong, Chun Yan; Wang, Tai

2013-01-01

Hectares of genetically modified (GM) crops have increased exponentially since 1996, when such crops began to be commercialized. GM biotechnology, together with conventional breeding, has become the main approach to improving agronomic traits of crops. However, people are concerned about the safety of GM crops, especially GM-derived food and feed. Many efforts have been made to evaluate the unintended effects caused by the introduction of exogenous genes. "Omics" techniques have advantages over targeted analysis in evaluating such crops because of their use of high-throughput screening. Proteins are key players in gene function and are directly involved in metabolism and cellular development or have roles as toxins, antinutrients, or allergens, which are essential for human health. Thus, proteomics can be expected to become one of the most useful tools in safety assessment. This review assesses the potential of proteomics in evaluating various GM crops. We further describe the challenges in ensuring homogeneity and sensitivity in detection techniques.

Proteomic Analysis Reveals Inflammation Modulation of κ/ι-Carrageenan Hexaoses in Lipopolysaccharide-Induced RAW264.7 Macrophages.

PubMed

Guo, Juanjuan; Chen, Jinghao; Lu, Xu; Guo, Zebin; Huang, Zhiwei; Zeng, Shaoxiao; Zhang, Yi; Zheng, Baodong

2018-05-09

κ/ι-Carrageenan hexaoses (κ/ι-neocarrahexaoses, KCO-4) are a type of carrageenan oligosaccharide that have a broad spectrum of bioactivities due to the presence of sulfate groups. However, the anti-inflammatory capacity of purified carrageenan oligosaccharides and the underlying mechanism has not been completely elucidated. The present study aimed to investigate inflammatory signaling modulation of KCO-4 in LPS-induced macrophages using a quantitative proteomic strategy. KCO-4 inhibited the oversecretion of inflammatory mediators (i.e., NO, TNF-α, IL-1β, IL-8, iNOS, and COX-2). KCO-4 treatment altered proteome profile, and metabolic processes in mitochondria were significantly disrupted. The IPA network analysis proposed that KCO-4 triggered the NF-κB signaling pathway-dependent anti-inflammation process through the inhibition of CD14/Rel@p50 in LPS-induced RAW264.7 macrophages. These data improve our understanding of the anti-inflammatory mechanism and contribute to exposure biomarker screening of κ-carrageenan oligosaccharides.

NITPICK: peak identification for mass spectrometry data.

PubMed

Renard, Bernhard Y; Kirchner, Marc; Steen, Hanno; Steen, Judith A J; Hamprecht, Fred A

2008-08-28

The reliable extraction of features from mass spectra is a fundamental step in the automated analysis of proteomic mass spectrometry (MS) experiments. This contribution proposes a sparse template regression approach to peak picking called NITPICK. NITPICK is a Non-greedy, Iterative Template-based peak PICKer that deconvolves complex overlapping isotope distributions in multicomponent mass spectra. NITPICK is based on fractional averaging, a novel extension to Senko's well-known averaging model, and on a modified version of sparse, non-negative least angle regression, for which a suitable, statistically motivated early stopping criterion has been derived. The strength of NITPICK is the deconvolution of overlapping mixture mass spectra. Extensive comparative evaluation has been carried out and results are provided for simulated and real-world data sets. NITPICK outperforms pepex, to date the only alternate, publicly available, non-greedy feature extraction routine. NITPICK is available as software package for the R programming language and can be downloaded from (http://hci.iwr.uni-heidelberg.de/mip/proteomics/).

Advances in targeted proteomics and applications to biomedical research

PubMed Central

Shi, Tujin; Song, Ehwang; Nie, Song; Rodland, Karin D.; Liu, Tao; Qian, Wei-Jun; Smith, Richard D.

2016-01-01

Targeted proteomics technique has emerged as a powerful protein quantification tool in systems biology, biomedical research, and increasing for clinical applications. The most widely used targeted proteomics approach, selected reaction monitoring (SRM), also known as multiple reaction monitoring (MRM), can be used for quantification of cellular signaling networks and preclinical verification of candidate protein biomarkers. As an extension to our previous review on advances in SRM sensitivity herein we review recent advances in the method and technology for further enhancing SRM sensitivity (from 2012 to present), and highlighting its broad biomedical applications in human bodily fluids, tissue and cell lines. Furthermore, we also review two recently introduced targeted proteomics approaches, parallel reaction monitoring (PRM) and data-independent acquisition (DIA) with targeted data extraction on fast scanning high-resolution accurate-mass (HR/AM) instruments. Such HR/AM targeted quantification with monitoring all target product ions addresses SRM limitations effectively in specificity and multiplexing; whereas when compared to SRM, PRM and DIA are still in the infancy with a limited number of applications. Thus, for HR/AM targeted quantification we focus our discussion on method development, data processing and analysis, and its advantages and limitations in targeted proteomics. Finally, general perspectives on the potential of achieving both high sensitivity and high sample throughput for large-scale quantification of hundreds of target proteins are discussed. PMID:27302376

The Hemolymph Proteome of Fed and Starved Drosophila Larvae

PubMed Central

Goetze, Sandra; Ahrens, Christian H.; Omasits, Ulrich; Marty, Florian; Simigdala, Nikiana; Meyer, Imke; Wollscheid, Bernd; Brunner, Erich; Hafen, Ernst; Lehner, Christian F.

2013-01-01

The co-operation of specialized organ systems in complex multicellular organisms depends on effective chemical communication. Thus, body fluids (like blood, lymph or intraspinal fluid) contain myriads of signaling mediators apart from metabolites. Moreover, these fluids are also of crucial importance for immune and wound responses. Compositional analyses of human body fluids are therefore of paramount diagnostic importance. Further improving their comprehensiveness should increase our understanding of inter-organ communication. In arthropods, which have trachea for gas exchange and an open circulatory system, the single dominating interstitial fluid is the hemolymph. Accordingly, a detailed analysis of hemolymph composition should provide an especially comprehensive picture of chemical communication and defense in animals. Therefore we used an extensive protein fractionation workflow in combination with a discovery-driven proteomic approach to map out the detectable protein composition of hemolymph isolated from Drosophila larvae. Combined mass spectrometric analysis revealed more than 700 proteins extending far beyond the previously known Drosophila hemolymph proteome. Moreover, by comparing hemolymph isolated from either fed or starved larvae, we provide initial provisional insights concerning compositional changes in response to nutritional state. Storage proteins in particular were observed to be strongly reduced by starvation. Our hemolymph proteome catalog provides a rich basis for data mining, as exemplified by our identification of potential novel cytokines, as well as for future quantitative analyses by targeted proteomics. PMID:23840627

The hemolymph proteome of fed and starved Drosophila larvae.

PubMed

Handke, Björn; Poernbacher, Ingrid; Goetze, Sandra; Ahrens, Christian H; Omasits, Ulrich; Marty, Florian; Simigdala, Nikiana; Meyer, Imke; Wollscheid, Bernd; Brunner, Erich; Hafen, Ernst; Lehner, Christian F

2013-01-01

The co-operation of specialized organ systems in complex multicellular organisms depends on effective chemical communication. Thus, body fluids (like blood, lymph or intraspinal fluid) contain myriads of signaling mediators apart from metabolites. Moreover, these fluids are also of crucial importance for immune and wound responses. Compositional analyses of human body fluids are therefore of paramount diagnostic importance. Further improving their comprehensiveness should increase our understanding of inter-organ communication. In arthropods, which have trachea for gas exchange and an open circulatory system, the single dominating interstitial fluid is the hemolymph. Accordingly, a detailed analysis of hemolymph composition should provide an especially comprehensive picture of chemical communication and defense in animals. Therefore we used an extensive protein fractionation workflow in combination with a discovery-driven proteomic approach to map out the detectable protein composition of hemolymph isolated from Drosophila larvae. Combined mass spectrometric analysis revealed more than 700 proteins extending far beyond the previously known Drosophila hemolymph proteome. Moreover, by comparing hemolymph isolated from either fed or starved larvae, we provide initial provisional insights concerning compositional changes in response to nutritional state. Storage proteins in particular were observed to be strongly reduced by starvation. Our hemolymph proteome catalog provides a rich basis for data mining, as exemplified by our identification of potential novel cytokines, as well as for future quantitative analyses by targeted proteomics.

Breast tumors educate stromal tissue with individualized but coordinated proteomic signatures

PubMed Central

Wang, Xuya; Mooradian, Arshag D.; Erdmann-Gilmore, Petra; Zhang, Qiang; Viner, Rosa; Davies, Sherri R.; Huang, Kuan-lin; Bomgarden, Ryan; Van Tine, Brian A.; Shao, Jieya; Ding, Li; Li, Shunqiang; Ellis, Matthew J.; Rogers, John C.; Townsend, R. Reid; Fenyö, David; Held, Jason M.

2017-01-01

Cancer forms specialized microenvironmental niches that promote local invasion and colonization. Engrafted patient-derived xenografts (PDXs) locally invade and colonize naïve stroma, while enabling unambiguous molecular discrimination of human proteins in the tumor from mouse proteins in the microenvironment. To characterize how patient breast tumors form a niche and educate naïve stroma, subcutaneous breast cancer PDXs were globally profiled using species-specific quantitative proteomics. Regulation of PDX stromal proteins by breast tumors was extensive, with thirty-five percent of the stromal proteome consistently altered by tumors across different animals and passages. Differentially regulated proteins in the stroma clustered into six signatures that included both known and novel contributors to tumor invasion and colonization. Stromal proteomes were coordinately regulated, though the sets of proteins altered by each tumor were highly distinct. Integrated analysis of tumor and stromal proteins, a comparison possible in xenograft models, indicated that the known hallmarks of cancer contribute pleiotropically to establishing and maintaining the tumor’s microenvironmental niche. Tumor education of the stroma is therefore an intrinsic property of breast tumors that is highly individualized, yet proceeds by consistent, non-random and defined tumor-promoting molecular alterations. PMID:28790197

Combining Capillary Electrophoresis with Mass Spectrometry for Applications in Proteomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Simpson, David C.; Smith, Richard D.

2005-04-01

Throughout the field of global proteomics, ranging from simple organism studies to human medical applications, the high sample complexity creates demands for improved separations and analysis techniques. Furthermore, with increased organism complexity, the correlation between proteome and genome becomes less certain due to extensive mRNA processing prior to translation. In this way, the same DNA sequence can potentially code for regions in a number of distinct proteins; quantitative differences in expression (or abundance) between these often-related species are of significant interest. Well-established proteomics techniques, which use genomic information to identify peptides that originate from protease digestion, often cannot easily distinguishmore » between such gene products; intact protein-level analyses are required to complete the picture, particularly for identifying post-translational modifications. While chromatographic techniques are currently better suited to peptide analysis, capillary electrophoresis (CE) in combination with mass spectrometry (MS) may become important for intact protein analysis. This review focuses on CE/MS instrumentation and techniques showing promise for such applications, highlighting those with greatest potential. Reference will also be made to developments relevant to peptide-level analyses for use in time- or sample-limited situations.« less

Proteome reference map and regulation network of neonatal rat cardiomyocyte

PubMed Central

Li, Zi-jian; Liu, Ning; Han, Qi-de; Zhang, You-yi

2011-01-01

Aim: To study and establish a proteome reference map and regulation network of neonatal rat cardiomyocyte. Methods: Cultured cardiomyocytes of neonatal rats were used. All proteins expressed in the cardiomyocytes were separated and identified by two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Biological networks and pathways of the neonatal rat cardiomyocytes were analyzed using the Ingenuity Pathway Analysis (IPA) program (www.ingenuity.com). A 2-DE database was made accessible on-line by Make2ddb package on a web server. Results: More than 1000 proteins were separated on 2D gels, and 148 proteins were identified. The identified proteins were used for the construction of an extensible markup language-based database. Biological networks and pathways were constructed to analyze the functions associate with cardiomyocyte proteins in the database. The 2-DE database of rat cardiomyocyte proteins can be accessed at http://2d.bjmu.edu.cn. Conclusion: A proteome reference map and regulation network of the neonatal rat cardiomyocytes have been established, which may serve as an international platform for storage, analysis and visualization of cardiomyocyte proteomic data. PMID:21841810

Display of a maize cDNA library on baculovirus infected insect cells.

PubMed

Meller Harel, Helene Y; Fontaine, Veronique; Chen, Hongying; Jones, Ian M; Millner, Paul A

2008-08-12

Maize is a good model system for cereal crop genetics and development because of its rich genetic heritage and well-characterized morphology. The sequencing of its genome is well advanced, and new technologies for efficient proteomic analysis are needed. Baculovirus expression systems have been used for the last twenty years to express in insect cells a wide variety of eukaryotic proteins that require complex folding or extensive posttranslational modification. More recently, baculovirus display technologies based on the expression of foreign sequences on the surface of Autographa californica (AcMNPV) have been developed. We investigated the potential of a display methodology for a cDNA library of maize young seedlings. We constructed a full-length cDNA library of young maize etiolated seedlings in the transfer vector pAcTMVSVG. The library contained a total of 2.5 x 10(5) independent clones. Expression of two known maize proteins, calreticulin and auxin binding protein (ABP1), was shown by western blot analysis of protein extracts from insect cells infected with the cDNA library. Display of the two proteins in infected insect cells was shown by selective biopanning using magnetic cell sorting and demonstrated proof of concept that the baculovirus maize cDNA display library could be used to identify and isolate proteins. The maize cDNA library constructed in this study relies on the novel technology of baculovirus display and is unique in currently published cDNA libraries. Produced to demonstrate proof of principle, it opens the way for the development of a eukaryotic in vivo display tool which would be ideally suited for rapid screening of the maize proteome for binding partners, such as proteins involved in hormone regulation or defence.

Metaproteomics: Harnessing the power of high performance mass spectrometry to identify the suite of proteins that control metabolic activities in microbial communities

PubMed Central

Hettich, Robert L.; Pan, Chongle; Chourey, Karuna; Giannone, Richard J.

2013-01-01

Summary The availability of extensive genome information for many different microbes, including unculturable species in mixed communities from environmental samples, has enabled systems-biology interrogation by providing a means to access genomic, transcriptomic, and proteomic information. To this end, metaproteomics exploits the power of high performance mass spectrometry for extensive characterization of the complete suite of proteins expressed by a microbial community in an environmental sample. PMID:23469896

Identification of Antibody Targets for Tuberculosis Serology using High-Density Nucleic Acid Programmable Protein Arrays*

PubMed Central

Song, Lusheng; Wallstrom, Garrick; Yu, Xiaobo; Hopper, Marika; Van Duine, Jennifer; Steel, Jason; Park, Jin; Wiktor, Peter; Kahn, Peter; Brunner, Al; Wilson, Douglas; Jenny-Avital, Elizabeth R.; Qiu, Ji; Labaer, Joshua; Magee, D. Mitchell; Achkar, Jacqueline M.

2017-01-01

Better and more diverse biomarkers for the development of simple point-of-care tests for active tuberculosis (TB), a clinically heterogeneous disease, are urgently needed. We generated a proteomic Mycobacterium tuberculosis (Mtb) High-Density Nucleic Acid Programmable Protein Array (HD-NAPPA) that used a novel multiplexed strategy for expedited high-throughput screening for antibody responses to the Mtb proteome. We screened sera from HIV uninfected and coinfected TB patients and controls (n = 120) from the US and South Africa (SA) using the multiplex HD-NAPPA for discovery, followed by deconvolution and validation through single protein HD-NAPPA with biologically independent samples (n = 124). We verified the top proteins with enzyme-linked immunosorbent assays (ELISA) using the original screening and validation samples (n = 244) and heretofore untested samples (n = 41). We identified 8 proteins with TB biomarker value; four (Rv0054, Rv0831c, Rv2031c and Rv0222) of these were previously identified in serology studies, and four (Rv0948c, Rv2853, Rv3405c, Rv3544c) were not known to elicit antibody responses. Using ELISA data, we created classifiers that could discriminate patients' TB status according to geography (US or SA) and HIV (HIV- or HIV+) status. With ROC curve analysis under cross validation, the classifiers performed with an AUC for US/HIV- at 0.807; US/HIV+ at 0.782; SA/HIV- at 0.868; and SA/HIV+ at 0.723. With this study we demonstrate a new platform for biomarker/antibody screening and delineate its utility to identify previously unknown immunoreactive proteins. PMID:28223349

Use of HCA in Subproteome-immunization and Screening of Hybridoma Supernatants to Define Distinct Antibody Binding Patterns

PubMed Central

Szafran, Adam T.; Mancini, Maureen G.; Nickerson, Jeffrey A.; Edwards, Dean P.; Mancini, Michael A.

2016-01-01

Understanding the properties and functions of complex biological systems depends upon knowing the proteins present and the interactions between them. Recent advances in mass spectrometry have given us greater insights into the participating proteomes, however, monoclonal antibodies remain key to understanding the structures, functions, locations and macromolecular interactions of the involved proteins. The traditional single immunogen method to produce monoclonal antibodies using hybridoma technology are time, resource and cost intensive, limiting the number of reagents that are available. Using a high content analysis screening approach, we have developed a method in which a complex mixture of proteins (e.g., subproteome) is used to generate a panel of monoclonal antibodies specific to a subproteome located in a defined subcellular compartment such as the nucleus. The immunofluorescent images in the primary hybridoma screen are analyzed using an automated processing approach and classified using a recursive partitioning forest classification model derived from images obtained from the Human Protein Atlas. Using an ammonium sulfate purified nuclear matrix fraction as an example of reverse proteomics, we identified 866 hybridoma supernatants with a positive immunofluorescent signal. Of those, 402 produced a nuclear signal from which patterns similar to known nuclear matrix associated proteins were identified. Detailed here is our method, the analysis techniques, and a discussion of the application to further in vivo antibody production. PMID:26521976

Use of HCA in subproteome-immunization and screening of hybridoma supernatants to define distinct antibody binding patterns.

PubMed

Szafran, Adam T; Mancini, Maureen G; Nickerson, Jeffrey A; Edwards, Dean P; Mancini, Michael A

2016-03-01

Understanding the properties and functions of complex biological systems depends upon knowing the proteins present and the interactions between them. Recent advances in mass spectrometry have given us greater insights into the participating proteomes, however, monoclonal antibodies remain key to understanding the structures, functions, locations and macromolecular interactions of the involved proteins. The traditional single immunogen method to produce monoclonal antibodies using hybridoma technology are time, resource and cost intensive, limiting the number of reagents that are available. Using a high content analysis screening approach, we have developed a method in which a complex mixture of proteins (e.g., subproteome) is used to generate a panel of monoclonal antibodies specific to a subproteome located in a defined subcellular compartment such as the nucleus. The immunofluorescent images in the primary hybridoma screen are analyzed using an automated processing approach and classified using a recursive partitioning forest classification model derived from images obtained from the Human Protein Atlas. Using an ammonium sulfate purified nuclear matrix fraction as an example of reverse proteomics, we identified 866 hybridoma supernatants with a positive immunofluorescent signal. Of those, 402 produced a nuclear signal from which patterns similar to known nuclear matrix associated proteins were identified. Detailed here is our method, the analysis techniques, and a discussion of the application to further in vivo antibody production. Copyright © 2015 Elsevier Inc. All rights reserved.

Comprehensive Proteomic Analysis of Human Milk-derived Extracellular Vesicles Unveils a Novel Functional Proteome Distinct from Other Milk Components.

PubMed

van Herwijnen, Martijn J C; Zonneveld, Marijke I; Goerdayal, Soenita; Nolte-'t Hoen, Esther N M; Garssen, Johan; Stahl, Bernd; Maarten Altelaar, A F; Redegeld, Frank A; Wauben, Marca H M

2016-11-01

Breast milk contains several macromolecular components with distinctive functions, whereby milk fat globules and casein micelles mainly provide nutrition to the newborn, and whey contains molecules that can stimulate the newborn's developing immune system and gastrointestinal tract. Although extracellular vesicles (EV) have been identified in breast milk, their physiological function and composition has not been addressed in detail. EV are submicron sized vehicles released by cells for intercellular communication via selectively incorporated lipids, nucleic acids, and proteins. Because of the difficulty in separating EV from other milk components, an in-depth analysis of the proteome of human milk-derived EV is lacking. In this study, an extensive LC-MS/MS proteomic analysis was performed of EV that had been purified from breast milk of seven individual donors using a recently established, optimized density-gradient-based EV isolation protocol. A total of 1963 proteins were identified in milk-derived EV, including EV-associated proteins like CD9, Annexin A5, and Flotillin-1, with a remarkable overlap between the different donors. Interestingly, 198 of the identified proteins are not present in the human EV database Vesiclepedia, indicating that milk-derived EV harbor proteins not yet identified in EV of different origin. Similarly, the proteome of milk-derived EV was compared with that of other milk components. For this, data from 38 published milk proteomic studies were combined in order to construct the total milk proteome, which consists of 2698 unique proteins. Remarkably, 633 proteins identified in milk-derived EV have not yet been identified in human milk to date. Interestingly, these novel proteins include proteins involved in regulation of cell growth and controlling inflammatory signaling pathways, suggesting that milk-derived EVs could support the newborn's developing gastrointestinal tract and immune system. Overall, this study provides an expansion of the whole milk proteome and illustrates that milk-derived EV are macromolecular components with a unique functional proteome. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Targeting human pathogenic bacteria by siderophores: A proteomics review.

PubMed

Ferreira, Daniela; Seca, Ana M L; C G A, Diana; Silva, Artur M S

2016-08-11

Human bacterial infections are still a major public health problem throughout the world. Therefore it is fundamental to understand how pathogenic bacteria interact with their human host and to develop more advanced drugs or vaccines in response to the increasing bacterial resistance. Since iron is essential to bacterial survival and growth inside the host tissues, these microorganisms have developed highly efficient iron-acquisition systems; the most common one involves the secretion of iron chelators into the extracellular environment, known as siderophores, and the corresponding siderophore-membrane receptors or transporters responsible for the iron uptake. In the past few decades, several biochemical methods and genetic screens have been employed to track down and identify these iron-scavenging molecules. However, compared with the previous "static" approaches, proteomic identification is revealing far more molecules through full protein mapping and becoming more rapid and selective, leading the scientific and medical community to consider standardizing proteomic tools for clinical biomarker detection of bacterial infectious diseases. In this review, we focus on human pathogenic Gram-negative bacteria and discuss the importance of siderophores in their virulence and the available proteomic strategies to identify siderophore-related proteins and their expression level under different growth conditions. The promising use of siderophore antibiotics to overcome bacterial resistance and the future of proteomics in the routine clinical care are also mentioned. Proteomic strategies to identify siderophore-related proteins and their expression level can be helpful to control and/or find a cure of infectious deseases especially if related with multidrug resistance. Siderophores are low-molecular-weight compounds produced by bacteria which can become clinical biomarkers and/or antibiotics used mainly in "Trojan horse" type strategies. Due to the above mention we think that the promising use of siderophore to overcome bacterial resistance and the future of proteomics in the routine clinical care is a hot topic that should be discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification of factors that function in Drosophila salivary gland cell death during development using proteomics

PubMed Central

McPhee, C K; Balgley, B M; Nelson, C; Hill, J H; Batlevi, Y; Fang, X; Lee, C S; Baehrecke, E H

2013-01-01

Proteasome inhibitors induce cell death and are used in cancer therapy, but little is known about the relationship between proteasome impairment and cell death under normal physiological conditions. Here, we investigate the relationship between proteasome function and larval salivary gland cell death during development in Drosophila. Drosophila larval salivary gland cells undergo synchronized programmed cell death requiring both caspases and autophagy (Atg) genes during development. Here, we show that ubiquitin proteasome system (UPS) function is reduced during normal salivary gland cell death, and that ectopic proteasome impairment in salivary gland cells leads to early DNA fragmentation and salivary gland condensation in vivo. Shotgun proteomic analyses of purified dying salivary glands identified the UPS as the top category of proteins enriched, suggesting a possible compensatory induction of these factors to maintain proteolysis during cell death. We compared the proteome following ectopic proteasome impairment to the proteome during developmental cell death in salivary gland cells. Proteins that were enriched in both populations of cells were screened for their function in salivary gland degradation using RNAi knockdown. We identified several factors, including trol, a novel gene CG11880, and the cop9 signalsome component cop9 signalsome 6, as required for Drosophila larval salivary gland degradation. PMID:22935612

Two-Dimensional Differential In-Gel Electrophoresis Proteomic Approaches Reveal Urine Candidate Biomarkers in Pediatric Obstructive Sleep Apnea

PubMed Central

Gozal, David; Jortani, Saeed; Snow, Ayelet B.; Kheirandish-Gozal, Leila; Bhattacharjee, Rakesh; Kim, Jinkwan; Capdevila, Oscar Sans

2009-01-01

Rationale: Sleep studies are laborious, expensive, inaccessible, and inconvenient for diagnosing obstructive sleep apnea (OSA) in children. Objectives: To examine whether the urinary proteome uncovers specific clusters that are differentially expressed in the urine of children with OSA. Methods: Two-dimensional differential in-gel electrophoresis (2D-DIGE) and mass spectrometry proteomics followed by validation with western blot of ELISA. Measurements and Main Results: Morning urine proteins from 60 children with polysomnographically confirmed OSA and from matched children with primary snoring (n = 30) and control subjects (n = 30) were assessed. A total of 16 proteins that are differentially expressed in OSA were identified, and 7 were confirmed by either immunoblots or ELISA. Among the latter, receiver–operator curve analyses of urinary concentrations of uromodulin, urocortin-3, orosomucoid-1, and kallikrein assigned favorable predictive properties to these proteins. Furthermore, combinatorial approaches indicated that the presence of values beyond the calculated cutoff concentrations for three or more of the proteins yielded a sensitivity of 95% and a specificity of 100%. Conclusions: Proteomic approaches reveal that pediatric OSA is associated with specific and consistent alterations in urinary concentrations of specific protein clusters. Future studies aiming to validate this approach as a screening method of habitually snoring children appears warranted. PMID:19797158

Proteomic Screening of Antigenic Proteins from the Hard Tick, Haemaphysalis longicornis (Acari: Ixodidae)

PubMed Central

Kim, Young-Ha; slam, Mohammad Saiful; You, Myung-Jo

2015-01-01

Proteomic tools allow large-scale, high-throughput analyses for the detection, identification, and functional investigation of proteome. For detection of antigens from Haemaphysalis longicornis, 1-dimensional electrophoresis (1-DE) quantitative immunoblotting technique combined with 2-dimensional electrophoresis (2-DE) immunoblotting was used for whole body proteins from unfed and partially fed female ticks. Reactivity bands and 2-DE immunoblotting were performed following 2-DE electrophoresis to identify protein spots. The proteome of the partially fed female had a larger number of lower molecular weight proteins than that of the unfed female tick. The total number of detected spots was 818 for unfed and 670 for partially fed female ticks. The 2-DE immunoblotting identified 10 antigenic spots from unfed females and 8 antigenic spots from partially fed females. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF) of relevant spots identified calreticulin, putative secreted WC salivary protein, and a conserved hypothetical protein from the National Center for Biotechnology Information and Swiss Prot protein sequence databases. These findings indicate that most of the whole body components of these ticks are non-immunogenic. The data reported here will provide guidance in the identification of antigenic proteins to prevent infestation and diseases transmitted by H. longicornis. PMID:25748713

Plants versus Fungi and Oomycetes: Pathogenesis, Defense and Counter-Defense in the Proteomics Era

PubMed Central

El Hadrami, Abdelbasset; El-Bebany, Ahmed F.; Yao, Zhen; Adam, Lorne R.; El Hadrami, Ismailx; Daayf, Fouad

2012-01-01

Plant-fungi and plant-oomycete interactions have been studied at the proteomic level for many decades. However, it is only in the last few years, with the development of new approaches, combined with bioinformatics data mining tools, gel staining, and analytical instruments, such as 2D-PAGE/nanoflow-LC-MS/MS, that proteomic approaches thrived. They allow screening and analysis, at the sub-cellular level, of peptides and proteins resulting from plants, pathogens, and their interactions. They also highlight post-translational modifications to proteins, e.g., glycosylation, phosphorylation or cleavage. However, many challenges are encountered during in planta studies aimed at stressing details of host defenses and fungal and oomycete pathogenicity determinants during interactions. Dissecting the mechanisms of such host-pathogen systems, including pathogen counter-defenses, will ensure a step ahead towards understanding current outcomes of interactions from a co-evolutionary point of view, and eventually move a step forward in building more durable strategies for management of diseases caused by fungi and oomycetes. Unraveling intricacies of more complex proteomic interactions that involve additional microbes, i.e., PGPRs and symbiotic fungi, which strengthen plant defenses will generate valuable information on how pathosystems actually function in nature, and thereby provide clues to solving disease problems that engender major losses in crops every year. PMID:22837691

Proteomics Analysis Reveals Abnormal Electron Transport and Excessive Oxidative Stress Cause Mitochondrial Dysfunction in Placental Tissues of Early-Onset Preeclampsia.

PubMed

Xu, Zhongwei; Jin, Xiaohan; Cai, Wei; Zhou, Maobin; Shao, Ping; Yang, Zhen; Fu, Rong; Cao, Jin; Liu, Yan; Yu, Fang; Fan, Rong; Zhang, Yan; Zou, Shuang; Zhou, Xin; Yang, Ning; Chen, Xu; Li, Yuming

2018-04-20

Early-onset preeclampsia (EOS-PE) refers to preeclampsia that occurred before 34 gestation weeks. This study is conducted to explore the relationship between mitochondrial dysfunction and the pathogenesis of EOS-PE using proteomic strategy. To identify altering expressed mitochondrial proteins between severe EOS-PE and healthy pregnancies, enrichment of mitochondria coupled with iTRAQ-based quantitative proteomic method is performed. Immunohistochemistry (IHC) and western blot are performed to detect the alteration of changing expression proteins, and confirmed the accuracy of proteomic results. A total of 1372 proteins were quantified and 132 altering expressed proteins were screened, including 86 downregulated expression proteins and 46 upregulated expression proteins (p < 0.05). Bioinformatics analysis showed that differentially expressed proteins participated in numerous biological processes, including oxidation-reduction process, respiratory electron transport chain, and oxidative phosphorylation. Especially, mitochondria-related molecules, PRDX2, PARK7, BNIP3, BCL2, PDHA1, SUCLG1, ACADM, and NDUFV1, are involved in energy-production process in the matrix and membrane of mitochondria. Results of the experiment show that abnormal electron transport, excessive oxidative stress, and mitochondrion disassembly might be the main cause of mitochondrial dysfunction, and is related to the pathogenesis of EOS-PE. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Plants versus fungi and oomycetes: pathogenesis, defense and counter-defense in the proteomics era.

PubMed

El Hadrami, Abdelbasset; El-Bebany, Ahmed F; Yao, Zhen; Adam, Lorne R; El Hadrami, Ismailx; Daayf, Fouad

2012-01-01

Plant-fungi and plant-oomycete interactions have been studied at the proteomic level for many decades. However, it is only in the last few years, with the development of new approaches, combined with bioinformatics data mining tools, gel staining, and analytical instruments, such as 2D-PAGE/nanoflow-LC-MS/MS, that proteomic approaches thrived. They allow screening and analysis, at the sub-cellular level, of peptides and proteins resulting from plants, pathogens, and their interactions. They also highlight post-translational modifications to proteins, e.g., glycosylation, phosphorylation or cleavage. However, many challenges are encountered during in planta studies aimed at stressing details of host defenses and fungal and oomycete pathogenicity determinants during interactions. Dissecting the mechanisms of such host-pathogen systems, including pathogen counter-defenses, will ensure a step ahead towards understanding current outcomes of interactions from a co-evolutionary point of view, and eventually move a step forward in building more durable strategies for management of diseases caused by fungi and oomycetes. Unraveling intricacies of more complex proteomic interactions that involve additional microbes, i.e., PGPRs and symbiotic fungi, which strengthen plant defenses will generate valuable information on how pathosystems actually function in nature, and thereby provide clues to solving disease problems that engender major losses in crops every year.

Comparative evaluation of saliva collection methods for proteome analysis.

PubMed

Golatowski, Claas; Salazar, Manuela Gesell; Dhople, Vishnu Mukund; Hammer, Elke; Kocher, Thomas; Jehmlich, Nico; Völker, Uwe

2013-04-18

Saliva collection devices are widely used for large-scale screening approaches. This study was designed to compare the suitability of three different whole-saliva collection approaches for subsequent proteome analyses. From 9 young healthy volunteers (4 women and 5 men) saliva samples were collected either unstimulated by passive drooling or stimulated using a paraffin gum or Salivette® (cotton swab). Saliva volume, protein concentration and salivary protein patterns were analyzed comparatively. Samples collected using paraffin gum showed the highest saliva volume (4.1±1.5 ml) followed by Salivette® collection (1.8±0.4 ml) and drooling (1.0±0.4 ml). Saliva protein concentrations (average 1145 μg/ml) showed no significant differences between the three sampling schemes. Each collection approach facilitated the identification of about 160 proteins (≥2 distinct peptides) per subject, but collection-method dependent variations in protein composition were observed. Passive drooling, paraffin gum and Salivette® each allows similar coverage of the whole saliva proteome, but the specific proteins observed depended on the collection approach. Thus, only one type of collection device should be used for quantitative proteome analysis in one experiment, especially when performing large-scale cross-sectional or multi-centric studies. Copyright © 2013 Elsevier B.V. All rights reserved.

Genome-wide screen uncovers novel pathways for tRNA processing and nuclear-cytoplasmic dynamics.

PubMed

Wu, Jingyan; Bao, Alicia; Chatterjee, Kunal; Wan, Yao; Hopper, Anita K

2015-12-15

Transfer ribonucleic acids (tRNAs) are essential for protein synthesis. However, key gene products involved in tRNA biogenesis and subcellular movement remain to be discovered. We conducted the first comprehensive unbiased analysis of the role of nearly an entire proteome in tRNA biology and describe 162 novel and 12 previously known Saccharomyces cerevisiae gene products that function in tRNA processing, turnover, and subcellular movement. tRNA nuclear export is of particular interest because it is essential, but the known tRNA exporters (Los1 [exportin-t] and Msn5 [exportin-5]) are unessential. We report that mutations of CRM1 (Exportin-1), MEX67/MTR2 (TAP/p15), and five nucleoporins cause accumulation of unspliced tRNA, a hallmark of defective tRNA nuclear export. CRM1 mutation genetically interacts with los1Δ and causes altered tRNA nuclear-cytoplasmic distribution. The data implicate roles for the protein and mRNA nuclear export machineries in tRNA nuclear export. Mutations of genes encoding actin cytoskeleton components and mitochondrial outer membrane proteins also cause accumulation of unspliced tRNA, likely due to defective splicing on mitochondria. Additional gene products, such as chromatin modification enzymes, have unanticipated effects on pre-tRNA end processing. Thus, this genome-wide screen uncovered putative novel pathways for tRNA nuclear export and extensive links between tRNA biology and other aspects of cell physiology. © 2015 Wu et al.; Published by Cold Spring Harbor Laboratory Press.

Genome-wide screen uncovers novel pathways for tRNA processing and nuclear–cytoplasmic dynamics

PubMed Central

Wu, Jingyan; Bao, Alicia; Chatterjee, Kunal; Wan, Yao; Hopper, Anita K.

2015-01-01

Transfer ribonucleic acids (tRNAs) are essential for protein synthesis. However, key gene products involved in tRNA biogenesis and subcellular movement remain to be discovered. We conducted the first comprehensive unbiased analysis of the role of nearly an entire proteome in tRNA biology and describe 162 novel and 12 previously known Saccharomyces cerevisiae gene products that function in tRNA processing, turnover, and subcellular movement. tRNA nuclear export is of particular interest because it is essential, but the known tRNA exporters (Los1 [exportin-t] and Msn5 [exportin-5]) are unessential. We report that mutations of CRM1 (Exportin-1), MEX67/MTR2 (TAP/p15), and five nucleoporins cause accumulation of unspliced tRNA, a hallmark of defective tRNA nuclear export. CRM1 mutation genetically interacts with los1Δ and causes altered tRNA nuclear–cytoplasmic distribution. The data implicate roles for the protein and mRNA nuclear export machineries in tRNA nuclear export. Mutations of genes encoding actin cytoskeleton components and mitochondrial outer membrane proteins also cause accumulation of unspliced tRNA, likely due to defective splicing on mitochondria. Additional gene products, such as chromatin modification enzymes, have unanticipated effects on pre-tRNA end processing. Thus, this genome-wide screen uncovered putative novel pathways for tRNA nuclear export and extensive links between tRNA biology and other aspects of cell physiology. PMID:26680305

Screening of the binding of small molecules to proteins by desorption electrospray ionization mass spectrometry combined with protein microarray.

PubMed

Yao, Chenxi; Wang, Tao; Zhang, Buqing; He, Dacheng; Na, Na; Ouyang, Jin

2015-11-01

The interaction between bioactive small molecule ligands and proteins is one of the important research areas in proteomics. Herein, a simple and rapid method is established to screen small ligands that bind to proteins. We designed an agarose slide to immobilize different proteins. The protein microarrays were allowed to interact with different small ligands, and after washing, the microarrays were screened by desorption electrospray ionization mass spectrometry (DESI MS). This method can be applied to screen specific protein binding ligands and was shown for seven proteins and 34 known ligands for these proteins. In addition, a high-throughput screening was achieved, with the analysis requiring approximately 4 s for one sample spot. We then applied this method to determine the binding between the important protein matrix metalloproteinase-9 (MMP-9) and 88 small compounds. The molecular docking results confirmed the MS results, demonstrating that this method is suitable for the rapid and accurate screening of ligands binding to proteins. Graphical Abstract ᅟ.

Screening for Pancreatic Cancer

PubMed Central

Brand, Randall E.

2007-01-01

Despite improvements in the clinical and surgical management of pancreatic cancer, limited strides have been made in the early detection of this highly lethal malignancy. The majority of localized pancreatic tumors are asymptomatic, and the recognized presenting symptoms of pancreatic adenocarcinoma are often vague and heterogeneous in nature. These factors, coupled with the lack of a sensitive and noninvasive screening method, have made population-based screening for pancreatic cancer impossible. Nevertheless, at least two large institutions have performed multimodality-screening protocols for individuals with high risk of pancreatic cancer based on genetic predisposition and strong family history. Abnormalities noted during these screening protocols prompted further investigation or surgery that resulted in the discovery of benign, potentially malignant, and malignant pancreatic lesions. In addition to ductal epithelial pancreatic intraepithelial neoplasia, greater sensitivity has recently been achieved in the identification and characterization of precancerous mucinous pancreatic tumors. Advancements in proteomics and DNA microarray technology may confirm serum-based biomarkers that could be incorporated into future screening algorithms for pancreatic cancer. PMID:21960811

UV irradiation-induced methionine oxidation in human skin keratins: Mass spectrometry-based non-invasive proteomic analysis.

PubMed

Lee, Seon Hwa; Matsushima, Keita; Miyamoto, Kohei; Oe, Tomoyuki

2016-02-05

Ultraviolet (UV) radiation is the major environmental factor that causes oxidative skin damage. Keratins are the main constituents of human skin and have been identified as oxidative target proteins. We have recently developed a mass spectrometry (MS)-based non-invasive proteomic methodology to screen oxidative modifications in human skin keratins. Using this methodology, UV effects on methionine (Met) oxidation in human skin keratins were investigated. The initial screening revealed that Met(259), Met(262), and Met(296) in K1 keratin were the most susceptible oxidation sites upon UVA (or UVB) irradiation of human tape-stripped skin. Subsequent liquid chromatography/electrospray ionization-MS and tandem MS analyses confirmed amino acid sequences and oxidation sites of tryptic peptides D(290)VDGAYMTK(298) (P1) and N(258)MQDMVEDYR(267) (P2). The relative oxidation levels of P1 and P2 increased in a time-dependent manner upon UVA irradiation. Butylated hydroxytoluene was the most effective antioxidant for artifactual oxidation of Met residues. The relative oxidation levels of P1 and P2 after UVA irradiation for 48 h corresponded to treatment with 100mM hydrogen peroxide for 15 min. In addition, Met(259) was oxidized by only UVA irradiation. The Met sites identified in conjunction with the current proteomic methodology can be used to evaluate skin damage under various conditions of oxidative stress. We demonstrated that the relative Met oxidation levels in keratins directly reflected UV-induced damages to human tape-stripped skin. Human skin proteins isolated by tape stripping were analyzed by MS-based non-invasive proteomic methodology. Met(259), Met(262), and Met(296) in K1 keratin were the most susceptible oxidation sites upon UV irradiation. Met(259) was oxidized by only UVA irradiation. Quantitative LC/ESI-SRM/MS analyses confirmed a time-dependent increase in the relative oxidation of target peptides (P1 and P2) containing these Met residues, upon UVA irradiation of isolated human skin. The relative oxidation levels of P1 and P2 along with the current proteomic methodology could be applied to the assessment of oxidative stress levels in skin after exposure to sunlight. Copyright © 2015 Elsevier B.V. All rights reserved.

Building high-quality assay libraries for targeted analysis of SWATH MS data.

PubMed

Schubert, Olga T; Gillet, Ludovic C; Collins, Ben C; Navarro, Pedro; Rosenberger, George; Wolski, Witold E; Lam, Henry; Amodei, Dario; Mallick, Parag; MacLean, Brendan; Aebersold, Ruedi

2015-03-01

Targeted proteomics by selected/multiple reaction monitoring (S/MRM) or, on a larger scale, by SWATH (sequential window acquisition of all theoretical spectra) MS (mass spectrometry) typically relies on spectral reference libraries for peptide identification. Quality and coverage of these libraries are therefore of crucial importance for the performance of the methods. Here we present a detailed protocol that has been successfully used to build high-quality, extensive reference libraries supporting targeted proteomics by SWATH MS. We describe each step of the process, including data acquisition by discovery proteomics, assertion of peptide-spectrum matches (PSMs), generation of consensus spectra and compilation of MS coordinates that uniquely define each targeted peptide. Crucial steps such as false discovery rate (FDR) control, retention time normalization and handling of post-translationally modified peptides are detailed. Finally, we show how to use the library to extract SWATH data with the open-source software Skyline. The protocol takes 2-3 d to complete, depending on the extent of the library and the computational resources available.

Discovery of a Chemical Probe Bisamide (CCT251236): An Orally Bioavailable Efficacious Pirin Ligand from a Heat Shock Transcription Factor 1 (HSF1) Phenotypic Screen

PubMed Central

2016-01-01

Phenotypic screens, which focus on measuring and quantifying discrete cellular changes rather than affinity for individual recombinant proteins, have recently attracted renewed interest as an efficient strategy for drug discovery. In this article, we describe the discovery of a new chemical probe, bisamide (CCT251236), identified using an unbiased phenotypic screen to detect inhibitors of the HSF1 stress pathway. The chemical probe is orally bioavailable and displays efficacy in a human ovarian carcinoma xenograft model. By developing cell-based SAR and using chemical proteomics, we identified pirin as a high affinity molecular target, which was confirmed by SPR and crystallography. PMID:28004573

Quantitative proteomic characterization of the lung extracellular matrix in chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis.

PubMed

Åhrman, Emma; Hallgren, Oskar; Malmström, Lars; Hedström, Ulf; Malmström, Anders; Bjermer, Leif; Zhou, Xiao-Hong; Westergren-Thorsson, Gunilla; Malmström, Johan

2018-03-01

Remodeling of the extracellular matrix (ECM) is a common feature in lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Here, we applied a sequential tissue extraction strategy to describe disease-specific remodeling of human lung tissue in disease, using end-stages of COPD and IPF. Our strategy was based on quantitative comparison of the disease proteomes, with specific focus on the matrisome, using data-independent acquisition and targeted data analysis (SWATH-MS). Our work provides an in-depth proteomic characterization of human lung tissue during impaired tissue remodeling. In addition, we show important quantitative and qualitative effects of the solubility of matrisome proteins. COPD was characterized by a disease-specific increase in ECM regulators, metalloproteinase inhibitor 3 (TIMP3) and matrix metalloproteinase 28 (MMP-28), whereas for IPF, impairment in cell adhesion proteins, such as collagen VI and laminins, was most prominent. For both diseases, we identified increased levels of proteins involved in the regulation of endopeptidase activity, with several proteins belonging to the serpin family. The established human lung quantitative proteome inventory and the construction of a tissue-specific protein assay library provides a resource for future quantitative proteomic analyses of human lung tissues. We present a sequential tissue extraction strategy to determine changes in extractability of matrisome proteins in end-stage COPD and IPF compared to healthy control tissue. Extensive quantitative analysis of the proteome changes of the disease states revealed altered solubility of matrisome proteins involved in ECM regulators and cell-ECM communication. The results highlight disease-specific remodeling mechanisms associated with COPD and IPF. Copyright © 2018 Elsevier B.V. All rights reserved.

Proteome and metabolome profiling of cytokinin action in Arabidopsis identifying both distinct and similar responses to cytokinin down- and up-regulation.

PubMed

Černý, Martin; Kuklová, Alena; Hoehenwarter, Wolfgang; Fragner, Lena; Novák, Ondrej; Rotková, Gabriela; Jedelsky, Petr L; Žáková, Katerina; Šmehilová, Mária; Strnad, Miroslav; Weckwerth, Wolfram; Brzobohaty, Bretislav

2013-11-01

In plants, numerous developmental processes are controlled by cytokinin (CK) levels and their ratios to levels of other hormones. While molecular mechanisms underlying the regulatory roles of CKs have been intensely researched, proteomic and metabolomic responses to CK deficiency are unknown. Transgenic Arabidopsis seedlings carrying inducible barley cytokinin oxidase/dehydrogenase (CaMV35S>GR>HvCKX2) and agrobacterial isopentenyl transferase (CaMV35S>GR>ipt) constructs were profiled to elucidate proteome- and metabolome-wide responses to down- and up-regulation of CK levels, respectively. Proteome profiling identified >1100 proteins, 155 of which responded to HvCKX2 and/or ipt activation, mostly involved in growth, development, and/or hormone and light signalling. The metabolome profiling covered 79 metabolites, 33 of which responded to HvCKX2 and/or ipt activation, mostly amino acids, carbohydrates, and organic acids. Comparison of the data sets obtained from activated CaMV35S>GR>HvCKX2 and CaMV35S>GR>ipt plants revealed unexpectedly extensive overlaps. Integration of the proteomic and metabolomic data sets revealed: (i) novel components of molecular circuits involved in CK action (e.g. ribosomal proteins); (ii) previously unrecognized links to redox regulation and stress hormone signalling networks; and (iii) CK content markers. The striking overlaps in profiles observed in CK-deficient and CK-overproducing seedlings might explain surprising previously reported similarities between plants with down- and up-regulated CK levels.

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

PubMed

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.

Stage-Specific Transcriptome and Proteome Analyses of the Filarial Parasite Onchocerca volvulus and Its Wolbachia Endosymbiont

PubMed Central

Bennuru, Sasisekhar; Cotton, James A.; Ribeiro, Jose M. C.; Grote, Alexandra; Harsha, Bhavana; Holroyd, Nancy; Mhashilkar, Amruta; Molina, Douglas M.; Randall, Arlo Z.; Shandling, Adam D.; Unnasch, Thomas R.; Ghedin, Elodie; Berriman, Matthew

2016-01-01

ABSTRACT Onchocerciasis (river blindness) is a neglected tropical disease that has been successfully targeted by mass drug treatment programs in the Americas and small parts of Africa. Achieving the long-term goal of elimination of onchocerciasis, however, requires additional tools, including drugs, vaccines, and biomarkers of infection. Here, we describe the transcriptome and proteome profiles of the major vector and the human host stages (L1, L2, L3, molting L3, L4, adult male, and adult female) of Onchocerca volvulus along with the proteome of each parasitic stage and of its Wolbachia endosymbiont (wOv). In so doing, we have identified stage-specific pathways important to the parasite’s adaptation to its human host during its early development. Further, we generated a protein array that, when screened with well-characterized human samples, identified novel diagnostic biomarkers of O. volvulus infection and new potential vaccine candidates. This immunomic approach not only demonstrates the power of this postgenomic discovery platform but also provides additional tools for onchocerciasis control programs. PMID:27881553

Identification of Abiotic Stress Protein Biomarkers by Proteomic Screening of Crop Cultivar Diversity

PubMed Central

Barkla, Bronwyn J.

2016-01-01

Modern day agriculture practice is narrowing the genetic diversity in our food supply. This may compromise the ability to obtain high yield under extreme climactic conditions, threatening food security for a rapidly growing world population. To identify genetic diversity, tolerance mechanisms of cultivars, landraces and wild relatives of major crops can be identified and ultimately exploited for yield improvement. Quantitative proteomics allows for the identification of proteins that may contribute to tolerance mechanisms by directly comparing protein abundance under stress conditions between genotypes differing in their stress responses. In this review, a summary is provided of the data accumulated from quantitative proteomic comparisons of crop genotypes/cultivars which present different stress tolerance responses when exposed to various abiotic stress conditions, including drought, salinity, high/low temperature, nutrient deficiency and UV-B irradiation. This field of research aims to identify molecular features that can be developed as biomarkers for crop improvement, however without accurate phenotyping, careful experimental design, statistical robustness and appropriate biomarker validation and verification it will be challenging to deliver what is promised. PMID:28248236

Identification of Abiotic Stress Protein Biomarkers by Proteomic Screening of Crop Cultivar Diversity.

PubMed

Barkla, Bronwyn J

2016-09-08

Modern day agriculture practice is narrowing the genetic diversity in our food supply. This may compromise the ability to obtain high yield under extreme climactic conditions, threatening food security for a rapidly growing world population. To identify genetic diversity, tolerance mechanisms of cultivars, landraces and wild relatives of major crops can be identified and ultimately exploited for yield improvement. Quantitative proteomics allows for the identification of proteins that may contribute to tolerance mechanisms by directly comparing protein abundance under stress conditions between genotypes differing in their stress responses. In this review, a summary is provided of the data accumulated from quantitative proteomic comparisons of crop genotypes/cultivars which present different stress tolerance responses when exposed to various abiotic stress conditions, including drought, salinity, high/low temperature, nutrient deficiency and UV-B irradiation. This field of research aims to identify molecular features that can be developed as biomarkers for crop improvement, however without accurate phenotyping, careful experimental design, statistical robustness and appropriate biomarker validation and verification it will be challenging to deliver what is promised.

Comparative proteomics analysis of Bacillus amyloliquefaciens SQR9 revealed the key proteins involved in in situ root colonization.

PubMed

Qiu, Meihua; Xu, Zhihui; Li, Xingxing; Li, Qing; Zhang, Nan; Shen, Qirong; Zhang, Ruifu

2014-12-05

Bacillus Amyloliquefaciens SQR9 is a well-investigated plant growth-promoting rhizobacteria with strong root colonization capability. To identify the key proteins involved in in situ root colonization and biofilm formation, the proteomic profiles of planktonic and root colonized SQR9 cells were compared. A total of 755 proteins were identified, of which 78 and 95 proteins were significantly increased and deceased, respectively, when SQR9 was colonized on the root. The proteins that were closely affiliated with the root colonization belonged to the functional categories of biocontrol, detoxification, biofilm formation, cell motility and chemotaxis, transport, and degradation of plant polysaccharides. A two-component system protein ResE was increased 100-fold when compared to the planktonic status; impairment of the resE gene postponed the formation of cell biofilm and decreased the root colonization capability, which may be regulated through the spo0A-sinI-yqxM pathway. The SQR9 proteomic data provide valuable clues for screening key proteins in the plant-rhizobacteria interaction.

Proteomic identification of altered cerebral proteins in the complex regional pain syndrome animal model.

PubMed

Nahm, Francis Sahngun; Park, Zee-Yong; Nahm, Sang-Soep; Kim, Yong Chul; Lee, Pyung Bok

2014-01-01

Complex regional pain syndrome (CRPS) is a rare but debilitating pain disorder. Although the exact pathophysiology of CRPS is not fully understood, central and peripheral mechanisms might be involved in the development of this disorder. To reveal the central mechanism of CRPS, we conducted a proteomic analysis of rat cerebrum using the chronic postischemia pain (CPIP) model, a novel experimental model of CRPS. After generating the CPIP animal model, we performed a proteomic analysis of the rat cerebrum using a multidimensional protein identification technology, and screened the proteins differentially expressed between the CPIP and control groups. Results. A total of 155 proteins were differentially expressed between the CPIP and control groups: 125 increased and 30 decreased; expressions of proteins related to cell signaling, synaptic plasticity, regulation of cell proliferation, and cytoskeletal formation were increased in the CPIP group. However, proenkephalin A, cereblon, and neuroserpin were decreased in CPIP group. Altered expression of cerebral proteins in the CPIP model indicates cerebral involvement in the pathogenesis of CRPS. Further study is required to elucidate the roles of these proteins in the development and maintenance of CRPS.

Proteomics Reveals Plastid- and Periplastid-Targeted Proteins in the Chlorarachniophyte Alga Bigelowiella natans

PubMed Central

Hopkins, Julia F.; Spencer, David F.; Laboissiere, Sylvie; Neilson, Jonathan A.D.; Eveleigh, Robert J.M.; Durnford, Dion G.; Gray, Michael W.; Archibald, John M.

2012-01-01

Chlorarachniophytes are unicellular marine algae with plastids (chloroplasts) of secondary endosymbiotic origin. Chlorarachniophyte cells retain the remnant nucleus (nucleomorph) and cytoplasm (periplastidial compartment, PPC) of the green algal endosymbiont from which their plastid was derived. To characterize the diversity of nucleus-encoded proteins targeted to the chlorarachniophyte plastid, nucleomorph, and PPC, we isolated plastid–nucleomorph complexes from the model chlorarachniophyte Bigelowiella natans and subjected them to high-pressure liquid chromatography-tandem mass spectrometry. Our proteomic analysis, the first of its kind for a nucleomorph-bearing alga, resulted in the identification of 324 proteins with 95% confidence. Approximately 50% of these proteins have predicted bipartite leader sequences at their amino termini. Nucleus-encoded proteins make up >90% of the proteins identified. With respect to biological function, plastid-localized light-harvesting proteins were well represented, as were proteins involved in chlorophyll biosynthesis. Phylogenetic analyses revealed that many, but by no means all, of the proteins identified in our proteomic screen are of apparent green algal ancestry, consistent with the inferred evolutionary origin of the plastid and nucleomorph in chlorarachniophytes. PMID:23221610

The iron-responsive microsomal proteome of Aspergillus fumigatus.

PubMed

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 (p<0.0001), extensive microsomal proteome 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 (p<0.05, fold change ≥ 2), with 96 proteins showing increased abundance and 135 with decreased abundance following iron limitation, including predicted siderophore transporters. Fluorescently labelled FSC was only sequestered following A. fumigatus growth under iron-limiting conditions. Interestingly, human sera exhibited significantly increased reactivity (p<0.0001) against microsomal protein extracts obtained following iron-deplete growth. The opportunistic fungal pathogen Aspergillus 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 siderophore as a 'Trojan horse' to potentiate the efficacy of anti-fungal drugs. Finally, in addition to revealing the Aspergillus-specific IgG reactivity in normal human sera against microsomal proteins, there appears to be a significantly increased reactivity against microsomal proteins obtained following iron-restricted growth. We hypothesise that iron-limiting environment in humans, which has evolved to nutritionally limit pathogen growth in vivo, may also alter the fungal microsomal proteome. Copyright © 2016 Elsevier B.V. All rights reserved.

Concurrent Label-Free Mass Spectrometric Analysis of Dystrophin Isoform Dp427 and the Myofibrosis Marker Collagen in Crude Extracts from mdx-4cv Skeletal Muscles

PubMed Central

Murphy, Sandra; Zweyer, Margit; Mundegar, Rustam R.; Henry, Michael; Meleady, Paula; Swandulla, Dieter; Ohlendieck, Kay

2015-01-01

The full-length dystrophin protein isoform of 427 kDa (Dp427), the absence of which represents the principal abnormality in X-linked muscular dystrophy, is difficult to identify and characterize by routine proteomic screening approaches of crude tissue extracts. This is probably related to its large molecular size, its close association with the sarcolemmal membrane, and its existence within a heterogeneous glycoprotein complex. Here, we used a careful extraction procedure to isolate the total protein repertoire from normal versus dystrophic mdx-4cv skeletal muscles, in conjunction with label-free mass spectrometry, and successfully identified Dp427 by proteomic means. In contrast to a considerable number of previous comparative studies of the total skeletal muscle proteome, using whole tissue proteomics we show here for the first time that the reduced expression of this membrane cytoskeletal protein is the most significant alteration in dystrophinopathy. This agrees with the pathobiochemical concept that the almost complete absence of dystrophin is the main defect in Duchenne muscular dystrophy and that the mdx-4cv mouse model of dystrophinopathy exhibits only very few revertant fibers. Significant increases in collagens and associated fibrotic marker proteins, such as fibronectin, biglycan, asporin, decorin, prolargin, mimecan, and lumican were identified in dystrophin-deficient muscles. The up-regulation of collagen in mdx-4cv muscles was confirmed by immunofluorescence microscopy and immunoblotting. Thus, this is the first mass spectrometric study of crude tissue extracts that puts the proteomic identification of dystrophin in its proper pathophysiological context. PMID:28248273

Recognition of the polycistronic nature of human genes is critical to understanding the genotype-phenotype relationship.

PubMed

Brunet, Marie A; Levesque, Sébastien A; Hunting, Darel J; Cohen, Alan A; Roucou, Xavier

2018-05-01

Technological advances promise unprecedented opportunities for whole exome sequencing and proteomic analyses of populations. Currently, data from genome and exome sequencing or proteomic studies are searched against reference genome annotations. This provides the foundation for research and clinical screening for genetic causes of pathologies. However, current genome annotations substantially underestimate the proteomic information encoded within a gene. Numerous studies have now demonstrated the expression and function of alternative (mainly small, sometimes overlapping) ORFs within mature gene transcripts. This has important consequences for the correlation of phenotypes and genotypes. Most alternative ORFs are not yet annotated because of a lack of evidence, and this absence from databases precludes their detection by standard proteomic methods, such as mass spectrometry. Here, we demonstrate how current approaches tend to overlook alternative ORFs, hindering the discovery of new genetic drivers and fundamental research. We discuss available tools and techniques to improve identification of proteins from alternative ORFs and finally suggest a novel annotation system to permit a more complete representation of the transcriptomic and proteomic information contained within a gene. Given the crucial challenge of distinguishing functional ORFs from random ones, the suggested pipeline emphasizes both experimental data and conservation signatures. The addition of alternative ORFs in databases will render identification less serendipitous and advance the pace of research and genomic knowledge. This review highlights the urgent medical and research need to incorporate alternative ORFs in current genome annotations and thus permit their inclusion in hypotheses and models, which relate phenotypes and genotypes. © 2018 Brunet et al.; Published by Cold Spring Harbor Laboratory Press.

Development of data representation standards by the human proteome organization proteomics standards initiative

PubMed Central

Albar, Juan Pablo; Binz, Pierre-Alain; Eisenacher, Martin; Jones, Andrew R; Mayer, Gerhard; Omenn, Gilbert S; Orchard, Sandra; Vizcaíno, Juan Antonio; Hermjakob, Henning

2015-01-01

Objective To describe the goals of the Proteomics Standards Initiative (PSI) of the Human Proteome Organization, the methods that the PSI has employed to create data standards, the resulting output of the PSI, lessons learned from the PSI’s evolution, and future directions and synergies for the group. Materials and Methods The PSI has 5 categories of deliverables that have guided the group. These are minimum information guidelines, data formats, controlled vocabularies, resources and software tools, and dissemination activities. These deliverables are produced via the leadership and working group organization of the initiative, driven by frequent workshops and ongoing communication within the working groups. Official standards are subjected to a rigorous document process that includes several levels of peer review prior to release. Results We have produced and published minimum information guidelines describing what information should be provided when making data public, either via public repositories or other means. The PSI has produced a series of standard formats covering mass spectrometer input, mass spectrometer output, results of informatics analysis (both qualitative and quantitative analyses), reports of molecular interaction data, and gel electrophoresis analyses. We have produced controlled vocabularies that ensure that concepts are uniformly annotated in the formats and engaged in extensive software development and dissemination efforts so that the standards can efficiently be used by the community. Conclusion In its first dozen years of operation, the PSI has produced many standards that have accelerated the field of proteomics by facilitating data exchange and deposition to data repositories. We look to the future to continue developing standards for new proteomics technologies and workflows and mechanisms for integration with other omics data types. Our products facilitate the translation of genomics and proteomics findings to clinical and biological phenotypes. The PSI website can be accessed at http://www.psidev.info. PMID:25726569

Proteomics and Metabolomics: Two Emerging Areas for Legume Improvement

PubMed Central

Ramalingam, Abirami; Kudapa, Himabindu; Pazhamala, Lekha T.; Weckwerth, Wolfram; Varshney, Rajeev K.

2015-01-01

The crop legumes such as chickpea, common bean, cowpea, peanut, pigeonpea, soybean, etc. are important sources of nutrition and contribute to a significant amount of biological nitrogen fixation (>20 million tons of fixed nitrogen) in agriculture. However, the production of legumes is constrained due to abiotic and biotic stresses. It is therefore imperative to understand the molecular mechanisms of plant response to different stresses and identify key candidate genes regulating tolerance which can be deployed in breeding programs. The information obtained from transcriptomics has facilitated the identification of candidate genes for the given trait of interest and utilizing them in crop breeding programs to improve stress tolerance. However, the mechanisms of stress tolerance are complex due to the influence of multi-genes and post-transcriptional regulations. Furthermore, stress conditions greatly affect gene expression which in turn causes modifications in the composition of plant proteomes and metabolomes. Therefore, functional genomics involving various proteomics and metabolomics approaches have been obligatory for understanding plant stress tolerance. These approaches have also been found useful to unravel different pathways related to plant and seed development as well as symbiosis. Proteome and metabolome profiling using high-throughput based systems have been extensively applied in the model legume species, Medicago truncatula and Lotus japonicus, as well as in the model crop legume, soybean, to examine stress signaling pathways, cellular and developmental processes and nodule symbiosis. Moreover, the availability of protein reference maps as well as proteomics and metabolomics databases greatly support research and understanding of various biological processes in legumes. Protein-protein interaction techniques, particularly the yeast two-hybrid system have been advantageous for studying symbiosis and stress signaling in legumes. In this review, several studies on proteomics and metabolomics in model and crop legumes have been discussed. Additionally, applications of advanced proteomics and metabolomics approaches have also been included in this review for future applications in legume research. The integration of these “omics” approaches will greatly support the identification of accurate biomarkers in legume smart breeding programs. PMID:26734026

Antibody Characterization Process | Office of Cancer Clinical Proteomics Research

Cancer.gov

The goal of the NCI's Antibody Characterization Program (ACP) is to have three monoclonal antibodies produced for each successfully expressed/purified recombinant antigen and one antibody per peptide (1 to 3 peptides per protein). To date, over 4000 clones have been screened before selecting the current 393 antibodies. They are winnowed down based on the projected end use of the antibody.

Applications and Extensions of pClust to Big Microbial Proteomic Data

ERIC Educational Resources Information Center

Lockwood, Svetlana

2016-01-01

The goal of biological sciences is to understand the biomolecular mechanics of living organisms. Proteins serve as the foundation for organisms functional analysis and sequence analysis has shown to be invaluable in answering questions about individual organisms. The first step in any sequence analysis is alignment and it is common that even…

Cabazitaxel overcomes cisplatin resistance in germ cell tumour cells.

PubMed

Gerwing, Mirjam; Jacobsen, Christine; Dyshlovoy, Sergey; Hauschild, Jessica; Rohlfing, Tina; Oing, Christoph; Venz, Simone; Oldenburg, Jan; Oechsle, Karin; Bokemeyer, Carsten; von Amsberg, Gunhild; Honecker, Friedemann

2016-09-01

Cisplatin-based chemotherapy is highly effective in metastasized germ cell tumours (GCT). However, 10-30 % of patients develop resistance to cisplatin, requiring salvage therapy. We investigated the in vitro activity of paclitaxel and the novel taxane cabazitaxel in cisplatin-sensitive and -resistant GCT cell lines. In vitro activity of paclitaxel and cabazitaxel was determined by proliferation assays, and mode of action of cabazitaxel was assessed by western blotting and two screening approaches, i.e. whole proteome analysis and a human apoptosis array. Activity of paclitaxel and cabazitaxel was not affected by cisplatin resistance, suggesting that there is no cross-resistance between these agents in vitro. Cabazitaxel treatment showed a strong inhibitory effect on colony formation capacity. Cabazitaxel induced classical apoptosis in all cell lines, reflected by cleavage of PARP and caspase 3, without inducing specific changes in the cell cycle distribution. Using the proteomic and human apoptosis array screening approaches, differential regulation of several proteins, including members of the bcl-2 family, was found, giving first insights into the mode of action of cabazitaxel in GCT. Cabazitaxel shows promising in vitro activity in GCT cells, independent of levels of cisplatin resistance.

IdentiPy: An Extensible Search Engine for Protein Identification in Shotgun Proteomics.

PubMed

Levitsky, Lev I; Ivanov, Mark V; Lobas, Anna A; Bubis, Julia A; Tarasova, Irina A; Solovyeva, Elizaveta M; Pridatchenko, Marina L; Gorshkov, Mikhail V

2018-06-18

We present an open-source, extensible search engine for shotgun proteomics. Implemented in Python programming language, IdentiPy shows competitive processing speed and sensitivity compared with the state-of-the-art search engines. It is equipped with a user-friendly web interface, IdentiPy Server, enabling the use of a single server installation accessed from multiple workstations. Using a simplified version of X!Tandem scoring algorithm and its novel "autotune" feature, IdentiPy outperforms the popular alternatives on high-resolution data sets. Autotune adjusts the search parameters for the particular data set, resulting in improved search efficiency and simplifying the user experience. IdentiPy with the autotune feature shows higher sensitivity compared with the evaluated search engines. IdentiPy Server has built-in postprocessing and protein inference procedures and provides graphic visualization of the statistical properties of the data set and the search results. It is open-source and can be freely extended to use third-party scoring functions or processing algorithms and allows customization of the search workflow for specialized applications.

Impact of a short-term exposure to spaceflight on the phenotype, genome, transcriptome and proteome of Escherichia coli

NASA Astrophysics Data System (ADS)

Li, Tianzhi; Chang, De; Xu, Huiwen; Chen, Jiapeng; Su, Longxiang; Guo, Yinghua; Chen, Zhenhong; Wang, Yajuan; Wang, Li; Wang, Junfeng; Fang, Xiangqun; Liu, Changting

2015-07-01

Escherichia coli (E. coli) is the most widely applied model organism in current biological science. As a widespread opportunistic pathogen, E. coli can survive not only by symbiosis with human, but also outside the host as well, which necessitates the evaluation of its response to the space environment. Therefore, to keep humans safe in space, it is necessary to understand how the bacteria respond to this environment. Despite extensive investigations for a few decades, the response of E. coli to the real space environment is still controversial. To better understand the mechanisms how E. coli overcomes harsh environments such as microgravity in space and to investigate whether these factors may induce pathogenic changes in E. coli that are potentially detrimental to astronauts, we conducted detailed genomics, transcriptomic and proteomic studies on E. coli that experienced 17 days of spaceflight. By comparing two flight strains LCT-EC52 and LCT-EC59 to a control strain LCT-EC106 that was cultured under the same temperature conditions on the ground, we identified metabolism changes, polymorphism changes, differentially expressed genes and proteins in the two flight strains. The flight strains differed from the control in the utilization of more than 30 carbon sources. Two single nucleotide polymorphisms (SNPs) and one deletion were identified in the flight strains. The expression level of more than 1000 genes altered in flight strains. Genes involved in chemotaxis, lipid metabolism and cell motility express differently. Moreover, the two flight strains also differed extensively from each other in terms of metabolism, transcriptome and proteome, indicating the impact of space environment on individual cells is heterogeneous and probably genotype-dependent. This study presents the first systematic profile of E. coli genome, transcriptome and proteome after spaceflight, which helps to elucidate the mechanism that controls the adaptation of microbes to the space environment.

Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization.

PubMed

Sarraf, Shireen A; Raman, Malavika; Guarani-Pereira, Virginia; Sowa, Mathew E; Huttlin, Edward L; Gygi, Steven P; Harper, J Wade

2013-04-18

The PARKIN ubiquitin ligase (also known as PARK2) and its regulatory kinase PINK1 (also known as PARK6), often mutated in familial early-onset Parkinson's disease, have central roles in mitochondrial homeostasis and mitophagy. Whereas PARKIN is recruited to the mitochondrial outer membrane (MOM) upon depolarization via PINK1 action and can ubiquitylate porin, mitofusin and Miro proteins on the MOM, the full repertoire of PARKIN substrates--the PARKIN-dependent ubiquitylome--remains poorly defined. Here we use quantitative diGly capture proteomics (diGly) to elucidate the ubiquitylation site specificity and topology of PARKIN-dependent target modification in response to mitochondrial depolarization. Hundreds of dynamically regulated ubiquitylation sites in dozens of proteins were identified, with strong enrichment for MOM proteins, indicating that PARKIN dramatically alters the ubiquitylation status of the mitochondrial proteome. Using complementary interaction proteomics, we found depolarization-dependent PARKIN association with numerous MOM targets, autophagy receptors, and the proteasome. Mutation of the PARKIN active site residue C431, which has been found mutated in Parkinson's disease patients, largely disrupts these associations. Structural and topological analysis revealed extensive conservation of PARKIN-dependent ubiquitylation sites on cytoplasmic domains in vertebrate and Drosophila melanogaster MOM proteins. These studies provide a resource for understanding how the PINK1-PARKIN pathway re-sculpts the proteome to support mitochondrial homeostasis.

Respiratory Proteomics Today: Are Technological Advances for the Identification of Biomarker Signatures Catching up with Their Promise? A Critical Review of the Literature in the Decade 2004-2013.

PubMed

Viglio, Simona; Stolk, Jan; Iadarola, Paolo; Giuliano, Serena; Luisetti, Maurizio; Salvini, Roberta; Fumagalli, Marco; Bardoni, Anna

2014-01-22

To improve the knowledge on a variety of severe disorders, research has moved from the analysis of individual proteins to the investigation of all proteins expressed by a tissue/organism. This global proteomic approach could prove very useful: (i) for investigating the biochemical pathways involved in disease; (ii) for generating hypotheses; or (iii) as a tool for the identification of proteins differentially expressed in response to the disease state. Proteomics has not been used yet in the field of respiratory research as extensively as in other fields, only a few reproducible and clinically applicable molecular markers, which can assist in diagnosis, having been currently identified. The continuous advances in both instrumentation and methodology, which enable sensitive and quantitative proteomic analyses in much smaller amounts of biological material than before, will hopefully promote the identification of new candidate biomarkers in this area. The aim of this report is to critically review the application over the decade 2004-2013 of very sophisticated technologies to the study of respiratory disorders. The observed changes in protein expression profiles from tissues/fluids of patients affected by pulmonary disorders opens the route for the identification of novel pathological mediators of these disorders.

Mass spectrometry-based targeted quantitative proteomics: achieving sensitive and reproducible detection of proteins.

PubMed

Boja, Emily S; Rodriguez, Henry

2012-04-01

Traditional shotgun proteomics used to detect a mixture of hundreds to thousands of proteins through mass spectrometric analysis, has been the standard approach in research to profile protein content in a biological sample which could lead to the discovery of new (and all) protein candidates with diagnostic, prognostic, and therapeutic values. In practice, this approach requires significant resources and time, and does not necessarily represent the goal of the researcher who would rather study a subset of such discovered proteins (including their variations or posttranslational modifications) under different biological conditions. In this context, targeted proteomics is playing an increasingly important role in the accurate measurement of protein targets in biological samples in the hope of elucidating the molecular mechanism of cellular function via the understanding of intricate protein networks and pathways. One such (targeted) approach, selected reaction monitoring (or multiple reaction monitoring) mass spectrometry (MRM-MS), offers the capability of measuring multiple proteins with higher sensitivity and throughput than shotgun proteomics. Developing and validating MRM-MS-based assays, however, is an extensive and iterative process, requiring a coordinated and collaborative effort by the scientific community through the sharing of publicly accessible data and datasets, bioinformatic tools, standard operating procedures, and well characterized reagents. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Systematic Characterization of the Murine Mitochondrial Proteome Using Functionally Validated Cardiac Mitochondria

PubMed Central

Zhang, Jun; Li, Xiaohai; Mueller, Michael; Wang, Yueju; Zong, Chenggong; Deng, Ning; Vondriska, Thomas M.; Liem, David A.; Yang, Jeong-In; Korge, Paavo; Honda, Henry; Weiss, James N.; Apweiler, Rolf; Ping, Peipei

2009-01-01

Mitochondria play essential roles in cardiac pathophysiology and the murine model has been extensively used to investigate cardiovascular diseases. In the present study, we characterized murine cardiac mitochondria using an LC/MS/MS approach. We extracted and purified cardiac mitochondria; validated their functionality to ensure the final preparation contains necessary components to sustain their normal function; and subjected these validated organelles to LC/MS/MS-based protein identification. A total of 940 distinct proteins were identified from murine cardiac mitochondria, among which, 480 proteins were not previously identified by major proteomic profiling studies. The 940 proteins consist of functional clusters known to support oxidative phosphorylation, metabolism and biogenesis. In addition, there are several other clusters--including proteolysis, protein folding, and reduction/oxidation signaling-which ostensibly represent previously under-appreciated tasks of cardiac mitochondria. Moreover, many identified proteins were found to occupy other subcellular locations, including cytoplasm, ER, and golgi, in addition to their presence in the mitochondria. These results provide a comprehensive picture of the murine cardiac mitochondrial proteome and underscore tissue- and species-specification. Moreover, the use of functionally intact mitochondria insures that the proteomic observations in this organelle are relevant to its normal biology and facilitates decoding the interplay between mitochondria and other organelles. PMID:18348319

Proteomic Analysis of Lyme Disease: Global Protein Comparison of Three Strains of Borrelia burgdorferi

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jacobs, Jon M.; Yang, Xiaohua; Luft, Benjamin J.

2005-04-01

The Borrelia burgdorferi spirochete is the causative agent of Lyme disease, the most common tick-borne disease in the United States. It has been studied extensively to help understand its pathogenicity of infection and how it can persist in different mammalian hosts. We report the proteomic analysis of the archetype B. burgdorferi B31 strain and two other strains (ND40, and JD-1) having different Borrelia pathotypes using strong cation exchange fractionation of proteolytic peptides followed by high-resolution, reversed phase capillary liquid chromatography coupled with ion trap tandem mass spectrometric (LC-MS/MS) analysis. Protein identification was facilitated by the availability of the complete B31more » genome sequence. A total of 665 Borrelia proteins were identified representing ~38 % coverage of the theoretical B31 proteome. A significant overlap was observed between the identified proteins in direct comparisons between any two strains (>72%), but distinct differences were observed among identified hypothetical and outer membrane proteins of the three strains. Such a concurrent proteomic overview of three Borrelia strains based upon only the B31 genome sequence is shown to provide significant insights into the presence or absence of specific proteins and a broad overall comparison among strains.« less

ProteoWizard: open source software for rapid proteomics tools development.

PubMed

Kessner, Darren; Chambers, Matt; Burke, Robert; Agus, David; Mallick, Parag

2008-11-01

The ProteoWizard software project provides a modular and extensible set of open-source, cross-platform tools and libraries. The tools perform proteomics data analyses; the libraries enable rapid tool creation by providing a robust, pluggable development framework that simplifies and unifies data file access, and performs standard proteomics and LCMS dataset computations. The library contains readers and writers of the mzML data format, which has been written using modern C++ techniques and design principles and supports a variety of platforms with native compilers. The software has been specifically released under the Apache v2 license to ensure it can be used in both academic and commercial projects. In addition to the library, we also introduce a rapidly growing set of companion tools whose implementation helps to illustrate the simplicity of developing applications on top of the ProteoWizard library. Cross-platform software that compiles using native compilers (i.e. GCC on Linux, MSVC on Windows and XCode on OSX) is available for download free of charge, at http://proteowizard.sourceforge.net. This website also provides code examples, and documentation. It is our hope the ProteoWizard project will become a standard platform for proteomics development; consequently, code use, contribution and further development are strongly encouraged.

Role of DNA Repair Factor Xeroderma Pigmentosum Protein Group C in Response to Replication Stress As Revealed by DNA Fragile Site Affinity Chromatography and Quantitative Proteomics.

PubMed

Beresova, Lucie; Vesela, Eva; Chamrad, Ivo; Voller, Jiri; Yamada, Masayuki; Furst, Tomas; Lenobel, Rene; Chroma, Katarina; Gursky, Jan; Krizova, Katerina; Mistrik, Martin; Bartek, Jiri

2016-12-02

Replication stress (RS) fuels genomic instability and cancer development and may contribute to aging, raising the need to identify factors involved in cellular responses to such stress. Here, we present a strategy for identification of factors affecting the maintenance of common fragile sites (CFSs), which are genomic loci that are particularly sensitive to RS and suffer from increased breakage and rearrangements in tumors. A DNA probe designed to match the high flexibility island sequence typical for the commonly expressed CFS (FRA16D) was used as specific DNA affinity bait. Proteins significantly enriched at the FRA16D fragment under normal and replication stress conditions were identified using stable isotope labeling of amino acids in cell culture-based quantitative mass spectrometry. The identified proteins interacting with the FRA16D fragment included some known CFS stabilizers, thereby validating this screening approach. Among the hits from our screen so far not implicated in CFS maintenance, we chose Xeroderma pigmentosum protein group C (XPC) for further characterization. XPC is a key factor in the DNA repair pathway known as global genomic nucleotide excision repair (GG-NER), a mechanism whose several components were enriched at the FRA16D fragment in our screen. Functional experiments revealed defective checkpoint signaling and escape of DNA replication intermediates into mitosis and the next generation of XPC-depleted cells exposed to RS. Overall, our results provide insights into an unexpected biological role of XPC in response to replication stress and document the power of proteomics-based screening strategies to elucidate mechanisms of pathophysiological significance.

DAnTE: a statistical tool for quantitative analysis of –omics data

DOE Office of Scientific and Technical Information (OSTI.GOV)

Polpitiya, Ashoka D.; Qian, Weijun; Jaitly, Navdeep

2008-05-03

DAnTE (Data Analysis Tool Extension) is a statistical tool designed to address challenges unique to quantitative bottom-up, shotgun proteomics data. This tool has also been demonstrated for microarray data and can easily be extended to other high-throughput data types. DAnTE features selected normalization methods, missing value imputation algorithms, peptide to protein rollup methods, an extensive array of plotting functions, and a comprehensive ANOVA scheme that can handle unbalanced data and random effects. The Graphical User Interface (GUI) is designed to be very intuitive and user friendly.

The impact of age on the art of mammography and how to adapt accordingly.

PubMed

Lake, B; Cielecki, L; Williams, S; Worrall, C; Metelko, M

2017-11-01

Breast cancer is increasingly a disease of the elderly, and combined with the NHS Breast Screening Extension means that more elderly patients are having mammography. Increasing age can make mammography more technically difficult. This is a technical note detailing the results of a local audit which may be of interest due to potential service implications. A retrospective audit of the first year of screening extension of The Shropshire Breast Screening Programme. Aims to collect data on patient demographics and describe the technical adaptations developed in Shropshire. Breast screening extension has increased by 2.5 times the number of women aged 70-74 screened, and doubled the overall numbers of women over 70 screened. Significantly more older patients are being screened to present technical challenges to a screening programme. Data was obtained from a month of screening showed that 29% of patients over 70 needed extra time for positioning. Reasons included 22% difficulty in obtaining adequate positioning and 15% needed a relative to aid with consent. In the Shropshire screening programme different technical adaptations have been developed and are key to ensuring adequate images. These include double appointments, two radiographers, thorough assessment, steeper angles, seated examinations, from-below imaging and pre-planning for subsequent screen. Significantly more older women are having breast screening due to the increasing incidence of breast cancer and the Breast Screening Programme extension. Increasing age can significantly increase time taken for adequate imaging and present technical challenges. Development of technical adaptations to art of mammography is key to achieve adequate images. Copyright © 2017 The College of Radiographers. Published by Elsevier Ltd. All rights reserved.

Discovery of novel drug targets and their functions using phenotypic screening of natural products.

PubMed

Chang, Junghwa; Kwon, Ho Jeong

2016-03-01

Natural products are valuable resources that provide a variety of bioactive compounds and natural pharmacophores in modern drug discovery. Discovery of biologically active natural products and unraveling their target proteins to understand their mode of action have always been critical hurdles for their development into clinical drugs. For effective discovery and development of bioactive natural products into novel therapeutic drugs, comprehensive screening and identification of target proteins are indispensable. In this review, a systematic approach to understanding the mode of action of natural products isolated using phenotypic screening involving chemical proteomics-based target identification is introduced. This review highlights three natural products recently discovered via phenotypic screening, namely glucopiericidin A, ecumicin, and terpestacin, as representative case studies to revisit the pivotal role of natural products as powerful tools in discovering the novel functions and druggability of targets in biological systems and pathological diseases of interest.

Open reading frames associated with cancer in the dark matter of the human genome.

PubMed

Delgado, Ana Paula; Brandao, Pamela; Chapado, Maria Julia; Hamid, Sheilin; Narayanan, Ramaswamy

2014-01-01

The uncharacterized proteins (open reading frames, ORFs) in the human genome offer an opportunity to discover novel targets for cancer. A systematic analysis of the dark matter of the human proteome for druggability and biomarker discovery is crucial to mining the genome. Numerous data mining tools are available to mine these ORFs to develop a comprehensive knowledge base for future target discovery and validation. Using the Genetic Association Database, the ORFs of the human dark matter proteome were screened for evidence of association with neoplasms. The Phenome-Genome Integrator tool was used to establish phenotypic association with disease traits including cancer. Batch analysis of the tools for protein expression analysis, gene ontology and motifs and domains was used to characterize the ORFs. Sixty-two ORFs were identified for neoplasm association. The expression Quantitative Trait Loci (eQTL) analysis identified thirteen ORFs related to cancer traits. Protein expression, motifs and domain analysis and genome-wide association studies verified the relevance of these OncoORFs in diverse tumors. The OncoORFs are also associated with a wide variety of human diseases and disorders. Our results link the OncoORFs to diverse diseases and disorders. This suggests a complex landscape of the uncharacterized proteome in human diseases. These results open the dark matter of the proteome to novel cancer target research. Copyright© 2014, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

SwissPalm: Protein Palmitoylation database.

PubMed

Blanc, Mathieu; David, Fabrice; Abrami, Laurence; Migliozzi, Daniel; Armand, Florence; Bürgi, Jérôme; van der Goot, Françoise Gisou

2015-01-01

Protein S-palmitoylation is a reversible post-translational modification that regulates many key biological processes, although the full extent and functions of protein S-palmitoylation remain largely unexplored. Recent developments of new chemical methods have allowed the establishment of palmitoyl-proteomes of a variety of cell lines and tissues from different species.  As the amount of information generated by these high-throughput studies is increasing, the field requires centralization and comparison of this information. Here we present SwissPalm ( http://swisspalm.epfl.ch), our open, comprehensive, manually curated resource to study protein S-palmitoylation. It currently encompasses more than 5000 S-palmitoylated protein hits from seven species, and contains more than 500 specific sites of S-palmitoylation. SwissPalm also provides curated information and filters that increase the confidence in true positive hits, and integrates predictions of S-palmitoylated cysteine scores, orthologs and isoform multiple alignments. Systems analysis of the palmitoyl-proteome screens indicate that 10% or more of the human proteome is susceptible to S-palmitoylation. Moreover, ontology and pathway analyses of the human palmitoyl-proteome reveal that key biological functions involve this reversible lipid modification. Comparative analysis finally shows a strong crosstalk between S-palmitoylation and other post-translational modifications. Through the compilation of data and continuous updates, SwissPalm will provide a powerful tool to unravel the global importance of protein S-palmitoylation.

SwissPalm: Protein Palmitoylation database

PubMed Central

Abrami, Laurence; Migliozzi, Daniel; Armand, Florence; Bürgi, Jérôme; van der Goot, Françoise Gisou

2015-01-01

Protein S-palmitoylation is a reversible post-translational modification that regulates many key biological processes, although the full extent and functions of protein S-palmitoylation remain largely unexplored. Recent developments of new chemical methods have allowed the establishment of palmitoyl-proteomes of a variety of cell lines and tissues from different species.  As the amount of information generated by these high-throughput studies is increasing, the field requires centralization and comparison of this information. Here we present SwissPalm ( http://swisspalm.epfl.ch), our open, comprehensive, manually curated resource to study protein S-palmitoylation. It currently encompasses more than 5000 S-palmitoylated protein hits from seven species, and contains more than 500 specific sites of S-palmitoylation. SwissPalm also provides curated information and filters that increase the confidence in true positive hits, and integrates predictions of S-palmitoylated cysteine scores, orthologs and isoform multiple alignments. Systems analysis of the palmitoyl-proteome screens indicate that 10% or more of the human proteome is susceptible to S-palmitoylation. Moreover, ontology and pathway analyses of the human palmitoyl-proteome reveal that key biological functions involve this reversible lipid modification. Comparative analysis finally shows a strong crosstalk between S-palmitoylation and other post-translational modifications. Through the compilation of data and continuous updates, SwissPalm will provide a powerful tool to unravel the global importance of protein S-palmitoylation. PMID:26339475

Data file of a deep proteome analysis of the prefrontal cortex in aged mice with progranulin deficiency or neuronal overexpression of progranulin.

PubMed

Heidler, Juliana; Hardt, Stefanie; Wittig, Ilka; Tegeder, Irmgard

2016-12-01

Progranulin deficiency is associated with neurodegeneration in humans and in mice. The mechanisms likely involve progranulin-promoted removal of protein waste via autophagy. We performed a deep proteomic screen of the pre-frontal cortex in aged (13-15 months) female progranulin-deficient mice (GRN -/- ) and mice with inducible neuron-specific overexpression of progranulin (SLICK-GRN-OE) versus the respective control mice. Proteins were extracted and analyzed per liquid chromatography/mass spectrometry (LC/MS) on a Thermo Scientific™ Q Exactive Plus equipped with an ultra-high performance liquid chromatography unit and a Nanospray Flex Ion-Source. Full Scan MS-data were acquired using Xcalibur and raw files were analyzed using the proteomics software Max Quant. The mouse reference proteome set from uniprot (June 2015) was used to identify peptides and proteins. The DiB data file is a reduced MaxQuant output and includes peptide and protein identification, accession numbers, protein and gene names, sequence coverage and label free quantification (LFQ) values of each sample. Differences in protein expression in genotypes are presented in "Progranulin overexpression in sensory neurons attenuates neuropathic pain in mice: Role of autophagy" (C. Altmann, S. Hardt, C. Fischer, J. Heidler, H.Y. Lim, A. Haussler, B. Albuquerque, B. Zimmer, C. Moser, C. Behrends, F. Koentgen, I. Wittig, M.H. Schmidt, A.M. Clement, T. Deller, I. Tegeder, 2016) [1].

Deciphering the Function of New Gonococcal Vaccine Antigens Using Phenotypic Microarrays

PubMed Central

Baarda, Benjamin I.; Emerson, Sarah; Proteau, Philip J.

2017-01-01

ABSTRACT The function and extracellular location of cell envelope proteins make them attractive candidates for developing vaccines against bacterial diseases, including challenging drug-resistant pathogens, such as Neisseria gonorrhoeae. A proteomics-driven reverse vaccinology approach has delivered multiple gonorrhea vaccine candidates; however, the biological functions of many of them remain to be elucidated. Herein, the functions of six gonorrhea vaccine candidates—NGO2121, NGO1985, NGO2054, NGO2111, NGO1205, and NGO1344—in cell envelope homeostasis were probed using phenotype microarrays under 1,056 conditions and a ΔbamE mutant (Δngo1780) as a reference of perturbed outer membrane integrity. Optimal growth conditions for an N. gonorrhoeae phenotype microarray assay in defined liquid medium were developed, which can be useful in other applications, including rapid and thorough antimicrobial susceptibility assessment. Our studies revealed 91 conditions having uniquely positive or negative effects on one of the examined mutants. A cluster analysis of 37 and 57 commonly beneficial and detrimental compounds, respectively, revealed three separate phenotype groups: NGO2121 and NGO1985; NGO1344 and BamE; and the trio of NGO1205, NGO2111, and NGO2054, with the last protein forming an independent branch of this cluster. Similar phenotypes were associated with loss of these vaccine candidates in the highly antibiotic-resistant WHO X strain. Based on their extensive sensitivity phenomes, NGO1985 and NGO2121 appear to be the most promising vaccine candidates. This study establishes the principle that phenotype microarrays can be successfully applied to a fastidious bacterial organism, such as N. gonorrhoeae. IMPORTANCE Innovative approaches are required to develop vaccines against prevalent and neglected sexually transmitted infections, such as gonorrhea. Herein, we have utilized phenotype microarrays in the first such investigation into Neisseria gonorrhoeae to probe the function of proteome-derived vaccine candidates in cell envelope homeostasis. Information gained from this screening can feed the vaccine candidate decision tree by providing insights into the roles these proteins play in membrane permeability, integrity, and overall N. gonorrhoeae physiology. The optimized screening protocol can be applied in investigations into the function of other hypothetical proteins of N. gonorrhoeae discovered in the expanding number of whole-genome sequences, in addition to revealing phenotypic differences between clinical and laboratory strains. PMID:28630127

Identification of Bacterial Factors Involved in Type 1 Fimbria Expression using an Escherichia coli K12 Proteome Chip*

PubMed Central

Chen, Yi-Wen; Teng, Ching-Hao; Ho, Yu-Hsuan; Jessica Ho, Tien Yu; Huang, Wen-Chun; Hashimoto, Masayuki; Chiang, I-Yuan; Chen, Chien-Sheng

2014-01-01

Type 1 fimbriae are filamentous structures on Escherichia coli. These structures are important adherence factors. Because binding to the host cells is the first step of infection, type 1 fimbria is an important virulence factor of pathogenic E. coli. Expression of type 1 fimbria is regulated by a phase variation in which each individual bacterium can alternate between fimbriated (phase-ON) and nonfimbriated (phase-OFF) states. The phase variation is regulated by the flipping of the 314-bp fimS fragment, which contains the promoter driving the expression of the genes required for the synthesis of type 1 fimbria. Thus, the bacterial proteins able to interact with fimS are likely to be involved in regulating the expression of type 1 fimbria. To identify novel type 1 fimbria-regulating factors, we used an E. coli K12 proteome chip to screen for the bacterial factors able to interact with a 602-bp DNA fragment containing fimS and its adjacent regions. The Spr protein was identified by the proteome chip-based screening and further confirmed to be able to interact with fimS by electrophoretic mobility shift assay. Deletion of spr in the neonatal meningitis E. coli strain RS218 significantly increased the ratio of the bacterial colonies that contained the type 1 fimbria phase-ON cells on agar plates. In addition, Spr interfered with the interactions of fimS with the site-specific recombinases, FimB and FimE, which are responsible for mediating the flipping of fimS. These results suggest that Spr is involved in the regulation of type 1 fimbria expression through direct interaction with the invertible element fimS. These findings facilitate our understanding of the regulation of type 1 fimbria. PMID:24692643

Diabetic retinopathy: Proteomic approaches to help the differential diagnosis and to understand the underlying molecular mechanisms.

PubMed

Csősz, Éva; Deák, Eszter; Kalló, Gergő; Csutak, Adrienne; Tőzsér, József

2017-01-06

Diabetic retinopathy is the most common diabetic eye disease and a leading cause of blindness among patients with diabetes. The appearance and the severity of the symptoms correlate with the duration of diabetes and poor blood glucose level management. Diabetic retinopathy is also categorized as a chronic low-level inflammatory disease; the high blood glucose level promotes the accumulation of the advanced glycation end products and leads to the stimulation of monocytes and macrophages. Examination of protein level alterations in tears using state-of the art proteomics techniques have identified several proteins as possible biomarkers for the different stages of the diabetic retinopathy. Some of the differentially expressed tear proteins have a role in the barrier function of tears linking the diabetic retinopathy with another eye complication of diabetes, namely the diabetic keratopathy resulting in impaired wound healing. Understanding the molecular events leading to the eye complications caused by hyperglycemia may help the identification of novel biomarkers as well as therapeutic targets in order to improve quality of life of diabetic patients. Diabetic retinopathy (DR), the leading cause of blindness among diabetic patients can develop without any serious symptoms therefore the early detection is crucial. Because of the increasing prevalence there is a high need for improved screening methods able to diagnose DR as soon as possible. The non-invasive collection and the relatively high protein concentration make the tear fluid a good source for biomarker discovery helping the early diagnosis. In this work we have reviewed the administration of advanced proteomics techniques used in tear biomarker studies and the identified biomarkers with potential to improve the already existing screening methods for DR detection. Copyright © 2016 Elsevier B.V. All rights reserved.

Combining comparative proteomics and molecular genetics uncovers regulators of synaptic and axonal stability and degeneration in vivo.

PubMed

Wishart, Thomas M; Rooney, Timothy M; Lamont, Douglas J; Wright, Ann K; Morton, A Jennifer; Jackson, Mandy; Freeman, Marc R; Gillingwater, Thomas H

2012-01-01

Degeneration of synaptic and axonal compartments of neurons is an early event contributing to the pathogenesis of many neurodegenerative diseases, but the underlying molecular mechanisms remain unclear. Here, we demonstrate the effectiveness of a novel "top-down" approach for identifying proteins and functional pathways regulating neurodegeneration in distal compartments of neurons. A series of comparative quantitative proteomic screens on synapse-enriched fractions isolated from the mouse brain following injury identified dynamic perturbations occurring within the proteome during both initiation and onset phases of degeneration. In silico analyses highlighted significant clustering of proteins contributing to functional pathways regulating synaptic transmission and neurite development. Molecular markers of degeneration were conserved in injury and disease, with comparable responses observed in synapse-enriched fractions isolated from mouse models of Huntington's disease (HD) and spinocerebellar ataxia type 5. An initial screen targeting thirteen degeneration-associated proteins using mutant Drosophila lines revealed six potential regulators of synaptic and axonal degeneration in vivo. Mutations in CALB2, ROCK2, DNAJC5/CSP, and HIBCH partially delayed injury-induced neurodegeneration. Conversely, mutations in DNAJC6 and ALDHA1 led to spontaneous degeneration of distal axons and synapses. A more detailed genetic analysis of DNAJC5/CSP mutants confirmed that loss of DNAJC5/CSP was neuroprotective, robustly delaying degeneration in axonal and synaptic compartments. Our study has identified conserved molecular responses occurring within synapse-enriched fractions of the mouse brain during the early stages of neurodegeneration, focused on functional networks modulating synaptic transmission and incorporating molecular chaperones, cytoskeletal modifiers, and calcium-binding proteins. We propose that the proteins and functional pathways identified in the current study represent attractive targets for developing therapeutics aimed at modulating synaptic and axonal stability and neurodegeneration in vivo.

Computational Biomarker Pipeline from Discovery to Clinical Implementation: Plasma Proteomic Biomarkers for Cardiac Transplantation

PubMed Central

Cohen Freue, Gabriela V.; Meredith, Anna; Smith, Derek; Bergman, Axel; Sasaki, Mayu; Lam, Karen K. Y.; Hollander, Zsuzsanna; Opushneva, Nina; Takhar, Mandeep; Lin, David; Wilson-McManus, Janet; Balshaw, Robert; Keown, Paul A.; Borchers, Christoph H.; McManus, Bruce; Ng, Raymond T.; McMaster, W. Robert

2013-01-01

Recent technical advances in the field of quantitative proteomics have stimulated a large number of biomarker discovery studies of various diseases, providing avenues for new treatments and diagnostics. However, inherent challenges have limited the successful translation of candidate biomarkers into clinical use, thus highlighting the need for a robust analytical methodology to transition from biomarker discovery to clinical implementation. We have developed an end-to-end computational proteomic pipeline for biomarkers studies. At the discovery stage, the pipeline emphasizes different aspects of experimental design, appropriate statistical methodologies, and quality assessment of results. At the validation stage, the pipeline focuses on the migration of the results to a platform appropriate for external validation, and the development of a classifier score based on corroborated protein biomarkers. At the last stage towards clinical implementation, the main aims are to develop and validate an assay suitable for clinical deployment, and to calibrate the biomarker classifier using the developed assay. The proposed pipeline was applied to a biomarker study in cardiac transplantation aimed at developing a minimally invasive clinical test to monitor acute rejection. Starting with an untargeted screening of the human plasma proteome, five candidate biomarker proteins were identified. Rejection-regulated proteins reflect cellular and humoral immune responses, acute phase inflammatory pathways, and lipid metabolism biological processes. A multiplex multiple reaction monitoring mass-spectrometry (MRM-MS) assay was developed for the five candidate biomarkers and validated by enzyme-linked immune-sorbent (ELISA) and immunonephelometric assays (INA). A classifier score based on corroborated proteins demonstrated that the developed MRM-MS assay provides an appropriate methodology for an external validation, which is still in progress. Plasma proteomic biomarkers of acute cardiac rejection may offer a relevant post-transplant monitoring tool to effectively guide clinical care. The proposed computational pipeline is highly applicable to a wide range of biomarker proteomic studies. PMID:23592955

Differential Occurrence of Interactions and Interaction Domains in Proteins Containing Homopolymeric Amino Acid Repeats

PubMed Central

Pelassa, Ilaria; Fiumara, Ferdinando

2015-01-01

Homopolymeric amino acids repeats (AARs), which are widespread in proteomes, have often been viewed simply as spacers between protein domains, or even as “junk” sequences with no obvious function but with a potential to cause harm upon expansion as in genetic diseases associated with polyglutamine or polyalanine expansions, including Huntington disease and cleidocranial dysplasia. A growing body of evidence indicates however that at least some AARs can form organized, functional protein structures, and can regulate protein function. In particular, certain AARs can mediate protein-protein interactions, either through homotypic AAR-AAR contacts or through heterotypic contacts with other protein domains. It is still unclear however, whether AARs may have a generalized, proteome-wide role in shaping protein-protein interaction networks. Therefore, we have undertaken here a bioinformatics screening of the human proteome and interactome in search of quantitative evidence of such a role. We first identified the sets of proteins that contain repeats of any one of the 20 amino acids, as well as control sets of proteins chosen at random in the proteome. We then analyzed the connectivity between the proteins of the AAR-containing protein sets and we compared it with that observed in the corresponding control networks. We find evidence for different degrees of connectivity in the different AAR-containing protein networks. Indeed, networks of proteins containing polyglutamine, polyglutamate, polyproline, and other AARs show significantly increased levels of connectivity, whereas networks containing polyleucine and other hydrophobic repeats show lower degrees of connectivity. Furthermore, we observed that numerous protein-protein, -nucleic acid, and -lipid interaction domains are significantly enriched in specific AAR protein groups. These findings support the notion of a generalized, combinatorial role of AARs, together with conventional protein interaction domains, in shaping the interaction networks of the human proteome, and define proteome-wide knowledge that may guide the informed biological exploration of the role of AARs in protein interactions. PMID:26734058

MASS SPECTROMETRY PROTEOMICS METHOD AS A RAPID SCREENING TOOL FOR BACTERIAL CONTAMINATION OF FOOD

DTIC Science & Technology

2017-06-01

1460 Rabih Jabbour RESEARCH AND TECHNOLOGY DIRECTORATE Havas, Karyn A. U.S. DEPARTMENT OF AGRICULTURE ANIMAL PLANT HEALTH INSPECTION SERVICES...coli O157:H7 (U.S. Department of Agriculture [USDA] strain 43895), Salmonella enterica serotype Newport (USDA strain 15480), Listeria monocytogenes...agar Sp specificity TSB trypticase soy broth USDA U.S. Department of Agriculture Xcorr correlation score DISTRIBUTION LIST The

Tear film proteome in age-related macular degeneration.

PubMed

Winiarczyk, Mateusz; Kaarniranta, Kai; Winiarczyk, Stanisław; Adaszek, Łukasz; Winiarczyk, Dagmara; Mackiewicz, Jerzy

2018-06-01

Age-related macular degeneration (AMD) is the main reason for blindness in elderly people in the developed countries. Current screening protocols have limitations in detecting the early signs of retinal degeneration. Therefore, it would be desirable to find novel biomarkers for early detection of AMD. Development of novel biomarkers would help in the prevention, diagnostics, and treatment of AMD. Proteomic analysis of tear film has shown promise in this research area. If an optimal set of biomarkers could be obtained from accessible body fluids, it would represent a reliable way to monitor disease progression and response to novel therapies. Tear films were collected on Schirmer strips from a total of 22 patients (8 with wet AMD, 6 with dry AMD, and 8 control individuals). 2D electrophoresis was used to separate tear film proteins prior to their identification with matrix-assisted laser desorption/ionization time of flight spectrometer (MALDI-TOF/TOF) and matching with functional databases. A total of 342 proteins were identified. Most of them were previously described in various proteomic studies concerning AMD. Shootin-1, histatin-3, fidgetin-like protein 1, SRC kinase signaling inhibitor, Graves disease carrier protein, actin cytoplasmic 1, prolactin-inducible protein 1, and protein S100-A7A were upregulated in the tear film samples isolated from AMD patients and were not previously linked with this disease in any proteomic analysis. The upregulated proteins supplement our current knowledge of AMD pathogenesis, providing evidence that certain specific proteins are expressed into the tear film in AMD. As far we are aware, this is the first study to have undertaken a comprehensive in-depth analysis of the human tear film proteome in AMD patients.

Comparative Proteomic Analysis of Whole-Gut Lavage Fluid and Pancreatic Juice Reveals a Less Invasive Method of Sampling Pancreatic Secretions

PubMed Central

Rocker, Jana M; Tan, Marcus C; Thompson, Lee W; Contreras, Carlo M; DiPalma, Jack A; Pannell, Lewis K

2016-01-01

OBJECTIVES: There are currently no reliable, non-invasive screening tests for pancreatic ductal adenocarcinoma. The fluid secreted from the pancreatic ductal system (“pancreatic juice”) has been well-studied as a potential source of cancer biomarkers. However, it is invasive to collect. We recently observed that the proteomic profile of intestinal effluent from the bowel in response to administration of an oral bowel preparation solution (also known as whole-gut lavage fluid, WGLF) contains large amounts of pancreas-derived proteins. We therefore hypothesized that the proteomic profile is similar to that of pancreatic juice. In this study, we compared the proteomic profiles of 77 patients undergoing routine colonoscopy with the profiles of 19 samples of pure pancreatic juice collected during surgery. METHODS: WGLF was collected from patients undergoing routine colonoscopy, and pancreatic juice was collected from patients undergoing pancreatic surgery. Protein was isolated from both samples using an optimized method and analyzed by LC-MS/MS. Identified proteins were compared between samples and groups to determine similarity of the two fluids. We then compared our results with literature reports of pancreatic juice-based studies to determine similarity. RESULTS: We found 104 proteins in our pancreatic juice samples, of which 90% were also found in our WGLF samples. The majority (67%) of the total proteins found in the WGLF were common to pancreatic juice, with intestine-specific proteins making up a smaller proportion. CONCLUSIONS: WGLF and pancreatic juice appear to have similar proteomic profiles. This supports the notion that WGLF is a non-invasive, surrogate bio-fluid for pancreatic juice. Further studies are required to further elucidate its role in the diagnosis of pancreatic cancer. PMID:27228405

“Exosomics”—A Review of Biophysics, Biology and Biochemistry of Exosomes With a Focus on Human Breast Milk

PubMed Central

de la Torre Gomez, Carolina; Goreham, Renee V.; Bech Serra, Joan J.; Nann, Thomas; Kussmann, Martin

2018-01-01

Exosomes are biomolecular nanostructures released from cells. They carry specific biomolecular information and are mainly researched for their exquisite properties as a biomarker source and delivery system. We introduce exosomes in the context of other extracellular vesicles, describe their biophysical isolation and characterisation and discuss their biochemical profiling. Motivated by our interest in early-life nutrition and health, and corresponding studies enrolling lactating mothers and their infants, we zoom into exosomes derived from human breast milk. We argue that these should be more extensively studied at proteomic and micronutrient profiling level, because breast milk exosomes provide a more specific window into breast milk quality from an immunological (proteomics) and nutritional (micronutrient) perspective. Such enhanced breast milk exosome profiling would thereby complement and enrich the more classical whole breast milk analysis and is expected to deliver more functional insights than the rather descriptive analysis of human milk, or larger fractions thereof, such as milk fat globule membrane. We substantiate our arguments by a bioinformatic analysis of two published proteomic data sets of human breast milk exosomes. PMID:29636770

"Exosomics"-A Review of Biophysics, Biology and Biochemistry of Exosomes With a Focus on Human Breast Milk.

PubMed

de la Torre Gomez, Carolina; Goreham, Renee V; Bech Serra, Joan J; Nann, Thomas; Kussmann, Martin

2018-01-01

Exosomes are biomolecular nanostructures released from cells. They carry specific biomolecular information and are mainly researched for their exquisite properties as a biomarker source and delivery system. We introduce exosomes in the context of other extracellular vesicles, describe their biophysical isolation and characterisation and discuss their biochemical profiling. Motivated by our interest in early-life nutrition and health, and corresponding studies enrolling lactating mothers and their infants, we zoom into exosomes derived from human breast milk. We argue that these should be more extensively studied at proteomic and micronutrient profiling level, because breast milk exosomes provide a more specific window into breast milk quality from an immunological (proteomics) and nutritional (micronutrient) perspective. Such enhanced breast milk exosome profiling would thereby complement and enrich the more classical whole breast milk analysis and is expected to deliver more functional insights than the rather descriptive analysis of human milk, or larger fractions thereof, such as milk fat globule membrane. We substantiate our arguments by a bioinformatic analysis of two published proteomic data sets of human breast milk exosomes.

Proteomic analysis of skeletal organic matrix from the stony coral Stylophora pistillata

PubMed Central

Drake, Jeana L.; Mass, Tali; Haramaty, Liti; Zelzion, Ehud; Bhattacharya, Debashish; Falkowski, Paul G.

2013-01-01

It has long been recognized that a suite of proteins exists in coral skeletons that is critical for the oriented precipitation of calcium carbonate crystals, yet these proteins remain poorly characterized. Using liquid chromatography-tandem mass spectrometry analysis of proteins extracted from the cell-free skeleton of the hermatypic coral, Stylophora pistillata, combined with a draft genome assembly from the cnidarian host cells of the same species, we identified 36 coral skeletal organic matrix proteins. The proteome of the coral skeleton contains an assemblage of adhesion and structural proteins as well as two highly acidic proteins that may constitute a unique coral skeletal organic matrix protein subfamily. We compared the 36 skeletal organic matrix protein sequences to genome and transcriptome data from three other corals, three additional invertebrates, one vertebrate, and three single-celled organisms. This work represents a unique extensive proteomic analysis of biomineralization-related proteins in corals from which we identify a biomineralization “toolkit,” an organic scaffold upon which aragonite crystals can be deposited in specific orientations to form a phenotypically identifiable structure. PMID:23431140

The proteome of the wool cuticle.

PubMed

Koehn, Henning; Clerens, Stefan; Deb-Choudhury, Santanu; Morton, James D; Dyer, Jolon M; Plowman, Jeffrey E

2010-06-04

The cuticle is responsible for important wool fiber characteristics such as handle and abrasion resistance, which impact on the fiber's performance in both interior and apparel textiles. The cuticle proteome, however, is not well understood due to the difficulty in isolating pure wool cuticle and its significant resistance to protein extraction, which is attributed to the presence of extensive disulfide and isopeptide cross-linking. We investigated the proteome of highly pure Merino wool cuticle using a combined strategy of chemical and enzymatic digestion and identified 108 proteins, including proteins responsible for a variety of cellular processes. The majority of identified proteins belonged to keratin and nonkeratin protein families known to play an important role in molecular assembly and cellular structure. Keratin-associated, intermediate filament and cytoskeletal keratin proteins were identified as the most prominent keratinous cuticular constituents, while histones, tubulins, and desmosomes were the key nonkeratin structural proteins. We conclude that a variety of proteins contribute to cuticle structure and fiber characteristics, and that the keratinous protein families of IFPs and KAPs represent the most important cuticular constituents.

Molecular targets of alcohol action: translational research for pharmacotherapy development and screening

PubMed Central

Gorini, Giorgio; Bell, Richard L.; Mayfield, R. Dayne

2016-01-01

Summary Alcohol abuse and dependence are multifaceted disorders with neurobiological, psychological, and environmental components. Research on other complex neuropsychiatric diseases suggests that genetically influenced intermediate characteristics affect the risk for heavy alcohol consumption and its consequences. Diverse therapeutic interventions can be developed through identification of reliable biomarkers for this disorder and new pharmacological targets for its treatment. Advances in the fields of genomics and proteomics offer a number of possible targets for the development of new therapeutic approaches. This brain-focused review highlights studies identifying neurobiological systems associated with these targets and possible pharmacotherapies, summarizing evidence from clinically relevant animal and human studies, as well as sketching improvements and challenges facing the fields of proteomics and genomics. Concluding thoughts on using results from these profiling technologies for medication development are also presented. PMID:21199775

Genes and pathways co-associated with the exposure to multiple drugs of abuse, including alcohol, amphetamine/methamphetamine, cocaine, marijuana, morphine, and/or nicotine: a review of proteomics analyses.

PubMed

Wang, Ju; Yuan, Wenji; Li, Ming D

2011-12-01

Drug addiction is a chronic neuronal disease. In recent years, proteomics technology has been widely used to assess the protein expression in the brain tissues of both animals and humans exposed to addictive drugs. Through this approach, a large number of proteins potentially involved in the etiology of drug addictions have been identified, which provide a valuable resource to study protein function, biochemical pathways, and networks related to the molecular mechanisms underlying drug dependence. In this article, we summarize the recent application of proteomics to profiling protein expression patterns in animal or human brain tissues after the administration of alcohol, amphetamine/methamphetamine, cocaine, marijuana, morphine/heroin/butorphanol, or nicotine. From available reports, we compiled a list of 497 proteins associated with exposure to one or more addictive drugs, with 160 being related to exposure to at least two abused drugs. A number of biochemical pathways and biological processes appear to be enriched among these proteins, including synaptic transmission and signaling pathways related to neuronal functions. The data included in this work provide a summary and extension of the proteomics studies on drug addiction. Furthermore, the proteins and biological processes highlighted here may provide valuable insight into the cellular activities and biological processes in neurons in the development of drug addiction.

Directed proteomic analysis of the human nucleolus.

PubMed

Andersen, Jens S; Lyon, Carol E; Fox, Archa H; Leung, Anthony K L; Lam, Yun Wah; Steen, Hanno; Mann, Matthias; Lamond, Angus I

2002-01-08

The nucleolus is a subnuclear organelle containing the ribosomal RNA gene clusters and ribosome biogenesis factors. Recent studies suggest it may also have roles in RNA transport, RNA modification, and cell cycle regulation. Despite over 150 years of research into nucleoli, many aspects of their structure and function remain uncharacterized. We report a proteomic analysis of human nucleoli. Using a combination of mass spectrometry (MS) and sequence database searches, including online analysis of the draft human genome sequence, 271 proteins were identified. Over 30% of the nucleolar proteins were encoded by novel or uncharacterized genes, while the known proteins included several unexpected factors with no previously known nucleolar functions. MS analysis of nucleoli isolated from HeLa cells in which transcription had been inhibited showed that a subset of proteins was enriched. These data highlight the dynamic nature of the nucleolar proteome and show that proteins can either associate with nucleoli transiently or accumulate only under specific metabolic conditions. This extensive proteomic analysis shows that nucleoli have a surprisingly large protein complexity. The many novel factors and separate classes of proteins identified support the view that the nucleolus may perform additional functions beyond its known role in ribosome subunit biogenesis. The data also show that the protein composition of nucleoli is not static and can alter significantly in response to the metabolic state of the cell.

Post-genomics of microsporidia, with emphasis on a model of minimal eukaryotic proteome: a review.

PubMed

Texier, Catherine; Brosson, Damien; El Alaoui, Hicham; Méténier, Guy; Vivarès, Christian P

2005-05-01

The genome sequence of the microsporidian parasite Encephalitozoon cuniculi Levaditi, Nicolau et Schoen, 1923 contains about 2,000 genes that are representative of a non-redundant potential proteome composed of 1,909 protein chains. The purpose of this review is to relate some advances in the characterisation of this proteome through bioinformatics and experimental approaches. The reduced diversity of the set of E. cuniculi proteins is perceptible in all the compilations of predicted domains, orthologs, families and superfamilies, available in several public databases. The phyletic patterns of orthologs for seven eukaryotic organisms support an extensive gene loss in the fungal clade, with additional deletions in E. cuniculi. Most microsporidial orthologs are the smallest ones among eukaryotes, justifying an interest in the use of these compacted proteins to better discriminate between essential and non-essential regions. The three components of the E. cuniculi mRNA capping apparatus have been especially well characterized and the three-dimensional structure of the cap methyltransferase has been elucidated following the crystallisation of the microsporidial enzyme Ecm1. So far, our mass spectrometry-based analyses of the E. cuniculi spore proteome has led to the identification of about 170 proteins, one-quarter of these having no clearly predicted function. Immunocytochemical studies are in progress to determine the subcellular localisation of microsporidia-specific proteins. Post-translational modifications such as phosphorylation and glycosylation are expected to be soon explored.

Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization

PubMed Central

Sarraf, Shireen A.; Raman, Malavika; Guarani-Pereira, Virginia; Sowa, Mathew E.; Huttlin, Edward L.; Gygi, Steven P.; Harper, J. Wade

2013-01-01

The PARKIN (PARK2) ubiquitin ligase and its regulatory kinase PINK1 (PARK6), often mutated in familial early onset Parkinson’s Disease (PD), play central roles in mitochondrial homeostasis and mitophagy.1–3 While PARKIN is recruited to the mitochondrial outer membrane (MOM) upon depolarization via PINK1 action and can ubiquitylate Porin, Mitofusin, and Miro proteins on the MOM,1,4–11 the full repertoire of PARKIN substrates – the PARKIN-dependent ubiquitylome - remains poorly defined. Here we employ quantitative diGLY capture proteomics12,13 to elucidate the ubiquitylation site-specificity and topology of PARKIN-dependent target modification in response to mitochondrial depolarization. Hundreds of dynamically regulated ubiquitylation sites in dozens of proteins were identified, with strong enrichment for MOM proteins, indicating that PARKIN dramatically alters the ubiquitylation status of the mitochondrial proteome. Using complementary interaction proteomics, we found depolarization-dependent PARKIN association with numerous MOM targets, autophagy receptors, and the proteasome. Mutation of PARKIN’s active site residue C431, which has been found mutated in PD patients, largely disrupts these associations. Structural and topological analysis revealed extensive conservation of PARKIN-dependent ubiquitylation sites on cytoplasmic domains in vertebrate and D. melanogaster MOM proteins. These studies provide a resource for understanding how the PINK1-PARKIN pathway re-sculpts the proteome to support mitochondrial homeostasis. PMID:23503661

An extensive library of surrogate peptides for all human proteins.

PubMed

Mohammed, Yassene; Borchers, Christoph H

2015-11-03

Selecting the most appropriate surrogate peptides to represent a target protein is a major component of experimental design in Multiple Reaction Monitoring (MRM). Our software PeptidePicker with its v-score remains distinctive in its approach of integrating information about the proteins, their tryptic peptides, and the suitability of these peptides for MRM that is available online in UniProtKB, NCBI's dbSNP, ExPASy, PeptideAtlas, PRIDE, and GPMDB. The scoring algorithm reflects our "best knowledge" for selecting candidate peptides for MRM, based on the uniqueness of the peptide in the targeted proteome, its physiochemical properties, and whether it has previously been observed. Here we present an updated approach where we have already compiled a list of all possible surrogate peptides of the human proteome. Using our stringent selection criteria, the list includes 165k suitable MRM peptides covering 17k proteins of the human reviewed proteins in UniProtKB. Compared to average of 2-4min per protein for retrieving and integrating the information, the precompiled list includes all peptides available instantly. This allows a more cohesive and faster design of a multiplexed MRM experiment and provides insights into evidence for a protein's existence. We will keep this list up-to-date as proteomics data repositories continue to grow. This article is part of a Special Issue entitled: Computational Proteomics. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparative Proteomics Reveals Novel Components at the Plasma Membrane of Differentiated HepaRG Cells and Different Distribution in Hepatocyte- and Biliary-Like Cells

PubMed Central

Woods, Alisa G.; Lazar, Catalin; Radu, Gabriel L.; Darie, Costel C.; Branza-Nichita, Norica

2013-01-01

Hepatitis B virus (HBV) is a human pathogen causing severe liver disease and eventually death. Despite important progress in deciphering HBV internalization, the early virus-cell interactions leading to infection are not known. HepaRG is a human bipotent liver cell line bearing the unique ability to differentiate towards a mixture of hepatocyte- and biliary-like cells. In addition to expressing metabolic functions normally found in liver, differentiated HepaRG cells support HBV infection in vitro, thus resembling cultured primary hepatocytes more than other hepatoma cells. Therefore, extensive characterization of the plasma membrane proteome from HepaRG cells would allow the identification of new cellular factors potentially involved in infection. Here we analyzed the plasma membranes of non-differentiated and differentiated HepaRG cells using nanoliquid chromatography-tandem mass spectrometry to identify the differences between the proteomes and the changes that lead to differentiation of these cells. We followed up on differentially-regulated proteins in hepatocytes- and biliary-like cells, focusing on Cathepsins D and K, Cyclophilin A, Annexin 1/A1, PDI and PDI A4/ERp72. Major differences between the two proteomes were found, including differentially regulated proteins, protein-protein interactions and intracellular localizations following differentiation. The results advance our current understanding of HepaRG differentiation and the unique properties of these cells. PMID:23977166

Hands-on workshops as an effective means of learning advanced technologies including genomics, proteomics and bioinformatics.

PubMed

Reisdorph, Nichole; Stearman, Robert; Kechris, Katerina; Phang, Tzu Lip; Reisdorph, Richard; Prenni, Jessica; Erle, David J; Coldren, Christopher; Schey, Kevin; Nesvizhskii, Alexey; Geraci, Mark

2013-12-01

Genomics and proteomics have emerged as key technologies in biomedical research, resulting in a surge of interest in training by investigators keen to incorporate these technologies into their research. At least two types of training can be envisioned in order to produce meaningful results, quality publications and successful grant applications: (1) immediate short-term training workshops and (2) long-term graduate education or visiting scientist programs. We aimed to fill the former need by providing a comprehensive hands-on training course in genomics, proteomics and informatics in a coherent, experimentally-based framework. This was accomplished through a National Heart, Lung, and Blood Institute (NHLBI)-sponsored 10-day Genomics and Proteomics Hands-on Workshop held at National Jewish Health (NJH) and the University of Colorado School of Medicine (UCD). The course content included comprehensive lectures and laboratories in mass spectrometry and genomics technologies, extensive hands-on experience with instrumentation and software, video demonstrations, optional workshops, online sessions, invited keynote speakers, and local and national guest faculty. Here we describe the detailed curriculum and present the results of short- and long-term evaluations from course attendees. Our educational program consistently received positive reviews from participants and had a substantial impact on grant writing and review, manuscript submissions and publications. Copyright © 2013. Production and hosting by Elsevier Ltd.

Comparative proteomics reveals novel components at the plasma membrane of differentiated HepaRG cells and different distribution in hepatocyte- and biliary-like cells.

PubMed

Petrareanu, Catalina; Macovei, Alina; Sokolowska, Izabela; Woods, Alisa G; Lazar, Catalin; Radu, Gabriel L; Darie, Costel C; Branza-Nichita, Norica

2013-01-01

Hepatitis B virus (HBV) is a human pathogen causing severe liver disease and eventually death. Despite important progress in deciphering HBV internalization, the early virus-cell interactions leading to infection are not known. HepaRG is a human bipotent liver cell line bearing the unique ability to differentiate towards a mixture of hepatocyte- and biliary-like cells. In addition to expressing metabolic functions normally found in liver, differentiated HepaRG cells support HBV infection in vitro, thus resembling cultured primary hepatocytes more than other hepatoma cells. Therefore, extensive characterization of the plasma membrane proteome from HepaRG cells would allow the identification of new cellular factors potentially involved in infection. Here we analyzed the plasma membranes of non-differentiated and differentiated HepaRG cells using nanoliquid chromatography-tandem mass spectrometry to identify the differences between the proteomes and the changes that lead to differentiation of these cells. We followed up on differentially-regulated proteins in hepatocytes- and biliary-like cells, focusing on Cathepsins D and K, Cyclophilin A, Annexin 1/A1, PDI and PDI A4/ERp72. Major differences between the two proteomes were found, including differentially regulated proteins, protein-protein interactions and intracellular localizations following differentiation. The results advance our current understanding of HepaRG differentiation and the unique properties of these cells.

Display technologies: application for the discovery of drug and gene delivery agents

PubMed Central

Sergeeva, Anna; Kolonin, Mikhail G.; Molldrem, Jeffrey J.; Pasqualini, Renata; Arap, Wadih

2007-01-01

Recognition of molecular diversity of cell surface proteomes in disease is essential for the development of targeted therapies. Progress in targeted therapeutics requires establishing effective approaches for high-throughput identification of agents specific for clinically relevant cell surface markers. Over the past decade, a number of platform strategies have been developed to screen polypeptide libraries for ligands targeting receptors selectively expressed in the context of various cell surface proteomes. Streamlined procedures for identification of ligand-receptor pairs that could serve as targets in disease diagnosis, profiling, imaging and therapy have relied on the display technologies, in which polypeptides with desired binding profiles can be serially selected, in a process called biopanning, based on their physical linkage with the encoding nucleic acid. These technologies include virus/phage display, cell display, ribosomal display, mRNA display and covalent DNA display (CDT), with phage display being by far the most utilized. The scope of this review is the recent advancements in the display technologies with a particular emphasis on molecular mapping of cell surface proteomes with peptide phage display. Prospective applications of targeted compounds derived from display libraries in the discovery of targeted drugs and gene therapy vectors are discussed. PMID:17123658

Study of cellular oncometabolism via multidimensional protein identification technology.

PubMed

Aukim-Hastie, Claire; Garbis, Spiros D

2014-01-01

Cellular proteomics is becoming a widespread clinical application, matching the definition of bench-to-bedside translation. Among various fields of investigation, this approach can be applied to the study of the metabolic alterations that accompany oncogenesis and tumor progression, which are globally referred to as oncometabolism. Here, we describe a multidimensional protein identification technology (MuDPIT)-based strategy that can be employed to study the cellular proteome of malignant cells and tissues. This method has previously been shown to be compatible with the reproducible, in-depth analysis of up to a thousand proteins in clinical samples. The possibility to employ this technique to study clinical specimens demonstrates its robustness. MuDPIT is advantageous as compared to other approaches because it is direct, highly sensitive, and reproducible, it provides high resolution with ultra-high mass accuracy, it allows for relative quantifications, and it is compatible with multiplexing (thus limiting costs).This method enables the direct assessment of the proteomic profile of neoplastic cells and tissues and could be employed in the near future as a high-throughput, rapid, quantitative, and cost-effective screening platform for clinical samples. © 2014 Elsevier Inc. All rights reserved.

Near Real-Time Processing of Proteomics Data Using Hadoop.

PubMed

Hillman, Chris; Ahmad, Yasmeen; Whitehorn, Mark; Cobley, Andy

2014-03-01

This article presents a near real-time processing solution using MapReduce and Hadoop. The solution is aimed at some of the data management and processing challenges facing the life sciences community. Research into genes and their product proteins generates huge volumes of data that must be extensively preprocessed before any biological insight can be gained. In order to carry out this processing in a timely manner, we have investigated the use of techniques from the big data field. These are applied specifically to process data resulting from mass spectrometers in the course of proteomic experiments. Here we present methods of handling the raw data in Hadoop, and then we investigate a process for preprocessing the data using Java code and the MapReduce framework to identify 2D and 3D peaks.

Proteomic analysis of Aspergillus fumigatus - clinical implications.

PubMed

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.

Integrated Molecular Characterization of Uterine Carcinosarcoma.

PubMed

Cherniack, Andrew D; Shen, Hui; Walter, Vonn; Stewart, Chip; Murray, Bradley A; Bowlby, Reanne; Hu, Xin; Ling, Shiyun; Soslow, Robert A; Broaddus, Russell R; Zuna, Rosemary E; Robertson, Gordon; Laird, Peter W; Kucherlapati, Raju; Mills, Gordon B; Weinstein, John N; Zhang, Jiashan; Akbani, Rehan; Levine, Douglas A

2017-03-13

We performed genomic, epigenomic, transcriptomic, and proteomic characterizations of uterine carcinosarcomas (UCSs). Cohort samples had extensive copy-number alterations and highly recurrent somatic mutations. Frequent mutations were found in TP53, PTEN, PIK3CA, PPP2R1A, FBXW7, and KRAS, similar to endometrioid and serous uterine carcinomas. Transcriptome sequencing identified a strong epithelial-to-mesenchymal transition (EMT) gene signature in a subset of cases that was attributable to epigenetic alterations at microRNA promoters. The range of EMT scores in UCS was the largest among all tumor types studied via The Cancer Genome Atlas. UCSs shared proteomic features with gynecologic carcinomas and sarcomas with intermediate EMT features. Multiple somatic mutations and copy-number alterations in genes that are therapeutic targets were identified. Copyright © 2017 Elsevier Inc. All rights reserved.

Protein Changes in Macrophages Induced by Plasma from Rats Exposed to 35-GHz Millimeter Waves

DTIC Science & Technology

2010-12-01

HumanEffectiveness Directorate, Air Force Research Laboratory, Brooks City-Base,Texas A macrophage assay and proteomic screening were used to...mW/cm2 until core temperature reached 41.0 8C. Two-dimensional polyacrylamide gel electrophoresis, image analysis, and Western blotting were used to...stimulation. Proteins of interest were identified using peptide mass fingerprinting. Compared to plasma from sham- exposed rats, plasma from

Hydrodynamic and Mass Transport Properties of Microfluidic Geometries

DTIC Science & Technology

2013-12-01

functions, known as proteomics, allows researchers to build a library that can be used to rapidly detect abnormal or diseased states. Protein...intake of toxins and disease vectors to tissues and cells, and therefore screening assays are crucial to clinical diagnostic identification of disease ...understanding of aggregation-mediated diseases (176, 177). 5.3 Preliminary Results A key step in the calculation of the surface viscosity from the shapes of

Matrilin-1 expression is increased in the vertebral column of Atlantic salmon (Salmo salar L.) individuals displaying spinal fusions.

PubMed

Pedersen, Mona E; Takle, Harald; Ytteborg, Elisabeth; Veiseth-Kent, Eva; Enersen, Grethe; Færgestad, Ellen; Baeverfjord, Grete; Hannesson, Kirsten O

2011-12-01

We have previously characterized the development of vertebral fusions induced by elevated water temperature in Atlantic salmon. Molecular markers of bone and cartilage development together with histology were used to understand the complex pathology and mechanism in the development of this spinal malformation. In this study, we wanted to use proteomics, a non-hypothetical approach to screen for possible new markers involved in the fusion process. Proteins extracted from non-deformed and fused vertebrae of Atlantic salmon were therefore compared by two-dimensional electrophoresis (2DE) and MALDI-TOF analysis. Data analysis of protein spots in the 2DE gels demonstrated matrilin-1, also named cartilage matrix protein, to be the most highly up-regulated protein in fused compared with non-deformed vertebrae. Furthermore, real-time PCR analysis showed strong up-regulation of matrilin-1 mRNA in fused vertebrae. Immunohistochemistry demonstrated induced matrilin-1 expression in trans-differentiating cells undergoing a metaplastic shift toward chondrocytes in fusing vertebrae, whereas abundant expression was demonstrated in cartilaginous tissue and chordocytes of both non-deformed and fused vertebrae. These results identifies matrilin-1 as a new interesting candidate in the fusion process, and ratify the use of proteomic as a valuable technique to screen for markers involved in vertebral pathogenesis.

Top-Down Proteomics and Direct Surface Sampling of Neonatal Dried Blood Spots: Diagnosis of Unknown Hemoglobin Variants

NASA Astrophysics Data System (ADS)

Edwards, Rebecca L.; Griffiths, Paul; Bunch, Josephine; Cooper, Helen J.

2012-11-01

We have previously shown that liquid microjunction surface sampling of dried blood spots coupled with high resolution top-down mass spectrometry may be used for screening of common hemoglobin variants HbS, HbC, and HbD. In order to test the robustness of the approach, we have applied the approach to unknown hemoglobin variants. Six neonatal dried blood spot samples that had been identified as variants, but which could not be diagnosed by current screening methods, were analyzed by direct surface sampling top-down mass spectrometry. Both collision-induced dissociation and electron transfer dissociation mass spectrometry were employed. Four of the samples were identified as β-chain variants: two were heterozygous Hb D-Iran, one was heterozygous Hb Headington, and one was heterozygous Hb J-Baltimore. The fifth sample was identified as the α-chain variant heterozygous Hb Phnom Penh. Analysis of the sixth sample suggested that it did not in fact contain a variant. Adoption of the approach in the clinic would require speed in both data collection and interpretation. To address that issue, we have compared manual data analysis with freely available data analysis software (ProsightPTM). The results demonstrate the power of top-down proteomics for hemoglobin variant analysis in newborn samples.

Mitochondrial Targets for Pharmacological Intervention in Human Disease

PubMed Central

2015-01-01

Over the past several years, mitochondrial dysfunction has been linked to an increasing number of human illnesses, making mitochondrial proteins (MPs) an ever more appealing target for therapeutic intervention. With 20% of the mitochondrial proteome (312 of an estimated 1500 MPs) having known interactions with small molecules, MPs appear to be highly targetable. Yet, despite these targeted proteins functioning in a range of biological processes (including induction of apoptosis, calcium homeostasis, and metabolism), very few of the compounds targeting MPs find clinical use. Recent work has greatly expanded the number of proteins known to localize to the mitochondria and has generated a considerable increase in MP 3D structures available in public databases, allowing experimental screening and in silico prediction of mitochondrial drug targets on an unprecedented scale. Here, we summarize the current literature on clinically active drugs that target MPs, with a focus on how existing drug targets are distributed across biochemical pathways and organelle substructures. Also, we examine current strategies for mitochondrial drug discovery, focusing on genetic, proteomic, and chemogenomic assays, and relevant model systems. As cell models and screening techniques improve, MPs appear poised to emerge as relevant targets for a wide range of complex human diseases, an eventuality that can be expedited through systematic analysis of MP function. PMID:25367773

Proteomics for the authentication of fish species.

PubMed

Mazzeo, Maria Fiorella; Siciliano, Rosa Anna

2016-09-16

Assessment of seafood authenticity and origin, mainly in the case of processed products (fillets, sticks, baby food) represents the crucial point to prevent fraudulent deceptions thus guaranteeing market transparency and consumers health. The most dangerous practice that jeopardies fish safety is intentional or unintentional mislabeling, originating from the substitution of valuable fish species with inferior ones. Conventional analytical methods for fish authentication are becoming inadequate to comply with the strict regulations issued by the European Union and with the increase of mislabeling due to the introduction on the market of new fish species and market globalization. This evidence prompts the development of high-throughput approaches suitable to identify unambiguous biomarkers of authenticity and screen a large number of samples with minimal time consumption. Proteomics provides suitable and powerful tools to investigate main aspects of food quality and safety and has given an important contribution in the field of biomarkers discovery applied to food authentication. This report describes the most relevant methods developed to assess fish identity and offers a perspective on their potential in the evaluation of fish quality and safety thus depicting the key role of proteomics in the authentication of fish species and processed products. The assessment of fishery products authenticity is a main issue in the control quality process as deceptive practices could imply severe health risks. Proteomics based methods could significantly contribute to detect falsification and frauds, thus becoming a reliable operative first-line testing resource in food authentication. Copyright © 2016 Elsevier B.V. All rights reserved.

New serum biomarkers for detection of tuberculosis using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry.

PubMed

Liu, Ji-Yan; Jin, Lei; Zhao, Meng-Yuan; Zhang, Xin; Liu, Chi-Bo; Zhang, Yu-Xiang; Li, Fu-Jian; Zhou, Jian-Min; Wang, Hua-Jun; Li, Ji-Cheng

2011-10-01

New technologies for the early detection of tuberculosis (TB) are urgently needed. Pathological changes within an organ might be reflected in proteomic patterns in serum. The aim of the present study was to screen for the potential protein biomarkers in serum for the diagnosis of TB using proteomic fingerprint technology. Proteomic fingerprint technology combining protein chips with surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) was used to profile the serum proteins from 50 patients with TB, 25 patients with lung disease other than TB, and 25 healthy volunteers. The protein fingerprint expression of all the serum samples and the resulting profiles between TB and control groups were analyzed with the Biomarker Wizard system. A total of 30 discriminating m/z peaks were detected that were related to TB (p<0.01). The model of biomarkers constructed by the Biomarker Patterns Software based on the three biomarkers (2024, 8007, and 8598 Da) generated excellent separation between the TB and control groups. The sensitivity was 84.0% and the specificity was 86.0%. Blind test data indicated a sensitivity of 80.0% and a specificity of 84.2%. The data suggested a potential application of SELDI-TOF MS as an effective technology to profile serum proteome, and with pattern analysis, a diagnostic model comprising three potential biomarkers was indicated to differentiate people with TB and healthy controls rapidly and precisely.

Uridine monophosphate kinase as potential target for tuberculosis: from target to lead identification.

PubMed

Arvind, Akanksha; Jain, Vaibhav; Saravanan, Parameswaran; Mohan, C Gopi

2013-12-01

Mycobacterium tuberculosis (Mtb) is a causative agent of tuberculosis (TB) disease, which has affected approximately 2 billion people worldwide. Due to the emergence of resistance towards the existing drugs, discovery of new anti-TB drugs is an important global healthcare challenge. To address this problem, there is an urgent need to identify new drug targets in Mtb. In the present study, the subtractive genomics approach has been employed for the identification of new drug targets against TB. Screening the Mtb proteome using the Database of Essential Genes (DEG) and human proteome resulted in the identification of 60 key proteins which have no eukaryotic counterparts. Critical analysis of these proteins using Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways database revealed uridine monophosphate kinase (UMPK) enzyme as a potential drug target for developing novel anti-TB drugs. Homology model of Mtb-UMPK was constructed for the first time on the basis of the crystal structure of E. coli-UMPK, in order to understand its structure-function relationships, and which would in turn facilitate to perform structure-based inhibitor design. Furthermore, the structural similarity search was carried out using physiological inhibitor UTP of Mtb-UMPK to virtually screen ZINC database. Retrieved hits were further screened by implementing several filters like ADME and toxicity followed by molecular docking. Finally, on the basis of the Glide docking score and the mode of binding, 6 putative leads were identified as inhibitors of this enzyme which can potentially emerge as future drugs for the treatment of TB.

Multiple biomarkers in molecular oncology. I. Molecular diagnostics applications in cervical cancer detection.

PubMed

Malinowski, Douglas P

2007-03-01

The screening for cervical carcinoma and its malignant precursors (cervical neoplasia) currently employs morphology-based detection methods (Papanicolaou [Pap] smear) in addition to the detection of high-risk human papillomavirus. The combination of the Pap smear with human papillomavirus testing has achieved significant improvements in sensitivity for the detection of cervical disease. Diagnosis of cervical neoplasia is dependent upon histology assessment of cervical biopsy specimens. Attempts to improve the specificity of cervical disease screening have focused on the investigation of molecular biomarkers for adjunctive use in combination with the Pap smear. Active research into the genomic and proteomic alterations that occur during human papillomavirus-induced neoplastic transformation have begun to characterize some of the basic mechanisms inherent to the disease process of cervical cancer development. This research continues to demonstrate the complexity of multiple genomic and proteomic alterations that accumulate during the tumorigenesis process. Despite this diversity, basic patterns of uncontrolled signal transduction, cell cycle deregulation, activation of DNA replication and altered extracellular matrix interactions are beginning to emerge as common features inherent to cervical cancer development. Some of these gene or protein expression alterations have been investigated as potential biomarkers for screening and diagnostics applications. The contribution of multiple gene alterations in the development of cervical cancer suggests that the application of multiple biomarker panels has the potential to develop clinically useful molecular diagnostics. In this review, the application of biomarkers for the improvement of sensitivity and specificity of the detection of cervical neoplasia within cytology specimens will be discussed.

Plasma-based proteomics reveals immune response, complement and coagulation cascades pathway shifts in heat-stressed lactating dairy cows.

PubMed

Min, Li; Cheng, Jianbo; Zhao, Shengguo; Tian, He; Zhang, Yangdong; Li, Songli; Yang, Hongjian; Zheng, Nan; Wang, Jiaqi

2016-09-02

Heat stress (HS) has an enormous economic impact on the dairy industry. In recent years, many researchers have investigated changes in the gene expression and metabolomics profiles in dairy cows caused by HS. However, the proteomics profiles of heat-stressed dairy cows have not yet been completely elucidated. We compared plasma proteomics from HS-free and heat-stressed dairy cows using an iTRAQ labeling approach. After the depletion of high abundant proteins in the plasma, 1472 proteins were identified. Of these, 85 proteins were differentially abundant in cows exposed to HS relative to HS-free. Database searches combined with GO and KEGG pathway enrichment analyses revealed that many components of the complement and coagulation cascades were altered in heat-stressed cows compared with HS-free cows. Of these, many factors in the complement system (including complement components C1, C3, C5, C6, C7, C8, and C9, complement factor B, and factor H) were down-regulated by HS, while components of the coagulation system (including coagulation factors, vitamin K-dependent proteins, and fibrinogens) were up-regulated by HS. In conclusion, our results indicate that HS decreases plasma levels of complement system proteins, suggesting that immune function is impaired in dairy cows exposed to HS. Though many aspects of heat stress (HS) have been extensively researched, relatively little is known about the proteomics profile changes that occur during heat exposure. In this work, we employed a proteomics approach to investigate differential abundance of plasma proteins in HS-free and heat-stressed dairy cows. Database searches combined with GO and KEGG pathway enrichment analyses revealed that HS resulted in a decrease in complement components, suggesting that heat-stressed dairy cows have impaired immune function. In addition, through integrative analyses of proteomics and previous metabolomics, we showed enhanced glycolysis, lipid metabolic pathway shifts, and nitrogen repartitioning in dairy cows exposed to HS. Our findings expand our current knowledge on the effects of HS on plasma proteomics in dairy cows and offer a new perspective for future research. Copyright © 2016 Elsevier B.V. All rights reserved.

Analysis of proteome response to the mobile phone radiation in two types of human primary endothelial cells.

PubMed

Nylund, Reetta; Kuster, Niels; Leszczynski, Dariusz

2010-10-18

Use of mobile phones has widely increased over the past decade. However, in spite of the extensive research, the question of potential health effects of the mobile phone radiation remains unanswered. We have earlier proposed, and applied, proteomics as a tool to study biological effects of the mobile phone radiation, using as a model human endothelial cell line EA.hy926. Exposure of EA.hy926 cells to 900 MHz GSM radiation has caused statistically significant changes in expression of numerous proteins. However, exposure of EA.hy926 cells to 1800 MHz GSM signal had only very small effect on cell proteome, as compared with 900 MHz GSM exposure. In the present study, using as model human primary endothelial cells, we have examined whether exposure to 1800 MHz GSM mobile phone radiation can affect cell proteome. Primary human umbilical vein endothelial cells and primary human brain microvascular endothelial cells were exposed for 1 hour to 1800 MHz GSM mobile phone radiation at an average specific absorption rate of 2.0 W/kg. The cells were harvested immediately after the exposure and the protein expression patterns of the sham-exposed and radiation-exposed cells were examined using two dimensional difference gel electrophoresis-based proteomics (2DE-DIGE). There were observed numerous differences between the proteomes of human umbilical vein endothelial cells and human brain microvascular endothelial cells (both sham-exposed). These differences are most likely representing physiological differences between endothelia in different vascular beds. However, the exposure of both types of primary endothelial cells to mobile phone radiation did not cause any statistically significant changes in protein expression. Exposure of primary human endothelial cells to the mobile phone radiation, 1800 MHz GSM signal for 1 hour at an average specific absorption rate of 2.0 W/kg, does not affect protein expression, when the proteomes were examined immediately after the end of the exposure and when the false discovery rate correction was applied to analysis. This observation agrees with our earlier study showing that the 1800 MHz GSM radiation exposure had only very limited effect on the proteome of human endothelial cell line EA.hy926, as compared with the effect of 900 MHz GSM radiation.

Targeted Data Extraction of the MS/MS Spectra Generated by Data-independent Acquisition: A New Concept for Consistent and Accurate Proteome Analysis*

PubMed Central

Gillet, Ludovic C.; Navarro, Pedro; Tate, Stephen; Röst, Hannes; Selevsek, Nathalie; Reiter, Lukas; Bonner, Ron; Aebersold, Ruedi

2012-01-01

Most proteomic studies use liquid chromatography coupled to tandem mass spectrometry to identify and quantify the peptides generated by the proteolysis of a biological sample. However, with the current methods it remains challenging to rapidly, consistently, reproducibly, accurately, and sensitively detect and quantify large fractions of proteomes across multiple samples. Here we present a new strategy that systematically queries sample sets for the presence and quantity of essentially any protein of interest. It consists of using the information available in fragment ion spectral libraries to mine the complete fragment ion maps generated using a data-independent acquisition method. For this study, the data were acquired on a fast, high resolution quadrupole-quadrupole time-of-flight (TOF) instrument by repeatedly cycling through 32 consecutive 25-Da precursor isolation windows (swaths). This SWATH MS acquisition setup generates, in a single sample injection, time-resolved fragment ion spectra for all the analytes detectable within the 400–1200 m/z precursor range and the user-defined retention time window. We show that suitable combinations of fragment ions extracted from these data sets are sufficiently specific to confidently identify query peptides over a dynamic range of 4 orders of magnitude, even if the precursors of the queried peptides are not detectable in the survey scans. We also show that queried peptides are quantified with a consistency and accuracy comparable with that of selected reaction monitoring, the gold standard proteomic quantification method. Moreover, targeted data extraction enables ad libitum quantification refinement and dynamic extension of protein probing by iterative re-mining of the once-and-forever acquired data sets. This combination of unbiased, broad range precursor ion fragmentation and targeted data extraction alleviates most constraints of present proteomic methods and should be equally applicable to the comprehensive analysis of other classes of analytes, beyond proteomics. PMID:22261725

Large-Scale Off-Target Identification Using Fast and Accurate Dual Regularized One-Class Collaborative Filtering and Its Application to Drug Repurposing.

PubMed

Lim, Hansaim; Poleksic, Aleksandar; Yao, Yuan; Tong, Hanghang; He, Di; Zhuang, Luke; Meng, Patrick; Xie, Lei

2016-10-01

Target-based screening is one of the major approaches in drug discovery. Besides the intended target, unexpected drug off-target interactions often occur, and many of them have not been recognized and characterized. The off-target interactions can be responsible for either therapeutic or side effects. Thus, identifying the genome-wide off-targets of lead compounds or existing drugs will be critical for designing effective and safe drugs, and providing new opportunities for drug repurposing. Although many computational methods have been developed to predict drug-target interactions, they are either less accurate than the one that we are proposing here or computationally too intensive, thereby limiting their capability for large-scale off-target identification. In addition, the performances of most machine learning based algorithms have been mainly evaluated to predict off-target interactions in the same gene family for hundreds of chemicals. It is not clear how these algorithms perform in terms of detecting off-targets across gene families on a proteome scale. Here, we are presenting a fast and accurate off-target prediction method, REMAP, which is based on a dual regularized one-class collaborative filtering algorithm, to explore continuous chemical space, protein space, and their interactome on a large scale. When tested in a reliable, extensive, and cross-gene family benchmark, REMAP outperforms the state-of-the-art methods. Furthermore, REMAP is highly scalable. It can screen a dataset of 200 thousands chemicals against 20 thousands proteins within 2 hours. Using the reconstructed genome-wide target profile as the fingerprint of a chemical compound, we predicted that seven FDA-approved drugs can be repurposed as novel anti-cancer therapies. The anti-cancer activity of six of them is supported by experimental evidences. Thus, REMAP is a valuable addition to the existing in silico toolbox for drug target identification, drug repurposing, phenotypic screening, and side effect prediction. The software and benchmark are available at https://github.com/hansaimlim/REMAP.

Large-Scale Off-Target Identification Using Fast and Accurate Dual Regularized One-Class Collaborative Filtering and Its Application to Drug Repurposing

PubMed Central

Poleksic, Aleksandar; Yao, Yuan; Tong, Hanghang; Meng, Patrick; Xie, Lei

2016-01-01

Target-based screening is one of the major approaches in drug discovery. Besides the intended target, unexpected drug off-target interactions often occur, and many of them have not been recognized and characterized. The off-target interactions can be responsible for either therapeutic or side effects. Thus, identifying the genome-wide off-targets of lead compounds or existing drugs will be critical for designing effective and safe drugs, and providing new opportunities for drug repurposing. Although many computational methods have been developed to predict drug-target interactions, they are either less accurate than the one that we are proposing here or computationally too intensive, thereby limiting their capability for large-scale off-target identification. In addition, the performances of most machine learning based algorithms have been mainly evaluated to predict off-target interactions in the same gene family for hundreds of chemicals. It is not clear how these algorithms perform in terms of detecting off-targets across gene families on a proteome scale. Here, we are presenting a fast and accurate off-target prediction method, REMAP, which is based on a dual regularized one-class collaborative filtering algorithm, to explore continuous chemical space, protein space, and their interactome on a large scale. When tested in a reliable, extensive, and cross-gene family benchmark, REMAP outperforms the state-of-the-art methods. Furthermore, REMAP is highly scalable. It can screen a dataset of 200 thousands chemicals against 20 thousands proteins within 2 hours. Using the reconstructed genome-wide target profile as the fingerprint of a chemical compound, we predicted that seven FDA-approved drugs can be repurposed as novel anti-cancer therapies. The anti-cancer activity of six of them is supported by experimental evidences. Thus, REMAP is a valuable addition to the existing in silico toolbox for drug target identification, drug repurposing, phenotypic screening, and side effect prediction. The software and benchmark are available at https://github.com/hansaimlim/REMAP. PMID:27716836

Construction and Screening of a Lentiviral Secretome Library.

PubMed

Liu, Tao; Jia, Panpan; Ma, Huailei; Reed, Sean A; Luo, Xiaozhou; Larman, H Benjamin; Schultz, Peter G

2017-06-22

Over 2,000 human proteins are predicted to be secreted, but the biological function of the many of these proteins is still unknown. Moreover, a number of these proteins may act as new therapeutic agents or be targets for the development of therapeutic antibodies. To further explore the extracellular proteome, we have developed a secretome-enriched open reading frame (ORF) library that can be readily screened for autocrine activity in cell-based phenotypic or reporter assays. Next-generation sequencing (NGS) and database analysis predict that the library contains approximately 900 ORFs encoding known secreted proteins (accounting for 77.8% of the library), as well as genes encoding potentially unknown secreted proteins. In a proof-of-principle study, human TF-1 cells were screened for proliferative factors, and the known cytokine GMCSF was identified as a dominant hit. This library offers a relatively low-cost and straightforward approach for functional autocrine screens of secreted proteins. Copyright © 2017 Elsevier Ltd. All rights reserved.

Quantitative Proteomic Profiling of Prostate Cancer Reveals a Role for miR-128 in Prostate Cancer*

PubMed Central

Khan, Amjad P.; Poisson, Laila M.; Bhat, Vadiraja B.; Fermin, Damian; Zhao, Rong; Kalyana-Sundaram, Shanker; Michailidis, George; Nesvizhskii, Alexey I.; Omenn, Gilbert S.; Chinnaiyan, Arul M.; Sreekumar, Arun

2010-01-01

Multiple, complex molecular events characterize cancer development and progression. Deciphering the molecular networks that distinguish organ-confined disease from metastatic disease may lead to the identification of biomarkers of cancer invasion and disease aggressiveness. Although alterations in gene expression have been extensively quantified during neoplastic progression, complementary analyses of proteomic changes have been limited. Here we interrogate the proteomic alterations in a cohort of 15 prostate-derived tissues that included five each from adjacent benign prostate, clinically localized prostate cancer, and metastatic disease from distant sites. The experimental strategy couples isobaric tags for relative and absolute quantitation with multidimensional liquid phase peptide fractionation followed by tandem mass spectrometry. Over 1000 proteins were quantified across the specimens and delineated into clinically localized and metastatic prostate cancer-specific signatures. Included in these class-specific profiles were both proteins that were known to be dysregulated during prostate cancer progression and new ones defined by this study. Enrichment analysis of the prostate cancer-specific proteomic signature, to gain insight into the functional consequences of these alterations, revealed involvement of miR-128-a/b regulation during prostate cancer progression. This finding was validated using real time PCR analysis for microRNA transcript levels in an independent set of 15 clinical specimens. miR-128 levels were elevated in benign prostate epithelial cell lines compared with invasive prostate cancer cells. Knockdown of miR-128 induced invasion in benign prostate epithelial cells, whereas its overexpression attenuated invasion in prostate cancer cells. Taken together, our profiles of the proteomic alterations of prostate cancer progression revealed miR-128 as a potentially important negative regulator of prostate cancer cell invasion. PMID:19955085

A Mouse to Human Search for Plasma Proteome Changes Associated with Pancreatic Tumor Development

PubMed Central

Faca, Vitor M; Song, Kenneth S; Wang, Hong; Zhang, Qing; Krasnoselsky, Alexei L; Newcomb, Lisa F; Plentz, Ruben R; Gurumurthy, Sushma; Redston, Mark S; Pitteri, Sharon J; Pereira-Faca, Sandra R; Ireton, Renee C; Katayama, Hiroyuki; Glukhova, Veronika; Phanstiel, Douglas; Brenner, Dean E; Anderson, Michelle A; Misek, David; Scholler, Nathalie; Urban, Nicole D; Barnett, Matt J; Edelstein, Cim; Goodman, Gary E; Thornquist, Mark D; McIntosh, Martin W; DePinho, Ronald A; Bardeesy, Nabeel; Hanash, Samir M

2008-01-01

Background The complexity and heterogeneity of the human plasma proteome have presented significant challenges in the identification of protein changes associated with tumor development. Refined genetically engineered mouse (GEM) models of human cancer have been shown to faithfully recapitulate the molecular, biological, and clinical features of human disease. Here, we sought to exploit the merits of a well-characterized GEM model of pancreatic cancer to determine whether proteomics technologies allow identification of protein changes associated with tumor development and whether such changes are relevant to human pancreatic cancer. Methods and Findings Plasma was sampled from mice at early and advanced stages of tumor development and from matched controls. Using a proteomic approach based on extensive protein fractionation, we confidently identified 1,442 proteins that were distributed across seven orders of magnitude of abundance in plasma. Analysis of proteins chosen on the basis of increased levels in plasma from tumor-bearing mice and corroborating protein or RNA expression in tissue documented concordance in the blood from 30 newly diagnosed patients with pancreatic cancer relative to 30 control specimens. A panel of five proteins selected on the basis of their increased level at an early stage of tumor development in the mouse was tested in a blinded study in 26 humans from the CARET (Carotene and Retinol Efficacy Trial) cohort. The panel discriminated pancreatic cancer cases from matched controls in blood specimens obtained between 7 and 13 mo prior to the development of symptoms and clinical diagnosis of pancreatic cancer. Conclusions Our findings indicate that GEM models of cancer, in combination with in-depth proteomic analysis, provide a useful strategy to identify candidate markers applicable to human cancer with potential utility for early detection. PMID:18547137

Proteomes and Ubiquitylomes Analysis Reveals the Involvement of Ubiquitination in Protein Degradation in Petunias1

PubMed Central

Liu, Juanxu; Wei, Qian; Wang, Rongmin; Yang, Weiyuan; Ma, Yueyue; Chen, Guoju

2017-01-01

Petal senescence is a complex programmed process. It has been demonstrated previously that treatment with ethylene, a plant hormone involved in senescence, can extensively alter transcriptome and proteome profiles in plants. However, little is known regarding the impact of ethylene on posttranslational modification (PTM) or the association between PTM and the proteome. Protein degradation is one of the hallmarks of senescence, and ubiquitination, a major PTM in eukaryotes, plays important roles in protein degradation. In this study, we first obtained reference petunia (Petunia hybrida) transcriptome data via RNA sequencing. Next, we quantitatively investigated the petunia proteome and ubiquitylome and the association between them in petunia corollas following ethylene treatment. In total, 51,799 unigenes, 3,606 proteins, and 2,270 ubiquitination sites were quantified 16 h after ethylene treatment. Treatment with ethylene resulted in 14,448 down-regulated and 6,303 up-regulated unigenes (absolute log2 fold change > 1 and false discovery rate < 0.001), 284 down-regulated and 233 up-regulated proteins, and 320 up-regulated and 127 down-regulated ubiquitination sites using a 1.5-fold threshold (P < 0.05), indicating that global ubiquitination levels increase during ethylene-mediated corolla senescence in petunia. Several putative ubiquitin ligases were up-regulated at the protein and transcription levels. Our results showed that the global proteome and ubiquitylome were negatively correlated and that ubiquitination could be involved in the degradation of proteins during ethylene-mediated corolla senescence in petunia. Ethylene regulates hormone signaling transduction pathways at both the protein and ubiquitination levels in petunia corollas. In addition, our results revealed that ethylene increases the ubiquitination levels of proteins involved in endoplasmic reticulum-associated degradation. PMID:27810942

Mild caloric restriction up-regulates the expression of prohibitin: A proteome study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takahashi, Shoko; Masuda, Junko; Shimagami, Hiroshi

2011-02-18

Research highlights: {yields} Proteomic analysis was performed to elucidate physiological alterations induced by mild CR. {yields} The results suggest good reproducibility and possibility to grasp the important response of CR. {yields} The increase in prohibitin abundance was observed in CR groups by proteomic analysis. {yields} We hypothesize that prohibitin might be involved in the longevity induced by CR. -- Abstract: Caloric restriction (CR) is well known to expand lifespan in a variety of species and to retard many age-related diseases. The effects of relatively mild CR on the proteome profile in relation to lifespan have not yet been reported, despitemore » the more extensive studies of the stricter CR conditions. Thus, the present study was conducted to elucidate the protein profiles in rat livers after mild CR for a relatively short time. Young growing rats were fed CR diets (10% and 30% CR) for 1 month. We performed the differential proteomic analysis of the rat livers using two-dimensional electrophoresis combined with MALDI-TOF mass spectrometry. The most remarkable protein among the differentially expressed proteins was found to be prohibitin, the abundance of which was increased by 30% CR. Prohibitin is a ubiquitously expressed protein shown to suppress cell proliferation and to be related to longevity. The increase in prohibitin was observed both in 10% and 30% CR by Western blot analysis. Furthermore, induction of AMP-activated kinase (AMPK) protein, related to the actions of prohibitin in promoting longevity, was observed. The increased prohibitin level in response to subtle CR suggests that this increase may be one of the early events leading to the expansion of lifespan in response to CR.« less

In-depth analysis of the thylakoid membrane proteome of Arabidopsis thaliana chloroplasts: new proteins, new functions, and a plastid proteome database.

PubMed

Friso, Giulia; Giacomelli, Lisa; Ytterberg, A Jimmy; Peltier, Jean-Benoit; Rudella, Andrea; Sun, Qi; Wijk, Klaas J van

2004-02-01

An extensive analysis of the Arabidopsis thaliana peripheral and integral thylakoid membrane proteome was performed by sequential extractions with salt, detergent, and organic solvents, followed by multidimensional protein separation steps (reverse-phase HPLC and one- and two-dimensional electrophoresis gels), different enzymatic and nonenzymatic protein cleavage techniques, mass spectrometry, and bioinformatics. Altogether, 154 proteins were identified, of which 76 (49%) were alpha-helical integral membrane proteins. Twenty-seven new proteins without known function but with predicted chloroplast transit peptides were identified, of which 17 (63%) are integral membrane proteins. These new proteins, likely important in thylakoid biogenesis, include two rubredoxins, a potential metallochaperone, and a new DnaJ-like protein. The data were integrated with our analysis of the lumenal-enriched proteome. We identified 83 out of 100 known proteins of the thylakoid localized photosynthetic apparatus, including several new paralogues and some 20 proteins involved in protein insertion, assembly, folding, or proteolysis. An additional 16 proteins are involved in translation, demonstrating that the thylakoid membrane surface is an important site for protein synthesis. The high coverage of the photosynthetic apparatus and the identification of known hydrophobic proteins with low expression levels, such as cpSecE, Ohp1, and Ohp2, indicate an excellent dynamic resolution of the analysis. The sequential extraction process proved very helpful to validate transmembrane prediction. Our data also were cross-correlated to chloroplast subproteome analyses by other laboratories. All data are deposited in a new curated plastid proteome database (PPDB) with multiple search functions (http://cbsusrv01.tc.cornell.edu/users/ppdb/). This PPDB will serve as an expandable resource for the plant community.

Gibberellin-Stimulation of Rhizome Elongation and Differential GA-Responsive Proteomic Changes in Two Grass Species

PubMed Central

Ma, Xiqing; Huang, Bingru

2016-01-01

Rapid and extensive rhizome development is a desirable trait for perennial grass growth and adaptation to environmental stresses. The objective of this study was to determine proteomic changes and associated metabolic pathways of gibberellin (GA) -regulation of rhizome elongation in two perennial grass species differing in rhizome development. Plants of a short-rhizome bunch-type tall fescue (TF; Festuca arundinacea; ‘BR’) and an extensive rhizomatous Kentucky bluegrass (KB; Poa pratensis; ‘Baron’) were treated with 10 μM GA3 in hydroponic culture in growth chambers. The average rhizome length in KB was significantly longer than that in TF regardless of GA3 treatment, and increased significantly with GA3 treatment, to a greater extent than that in TF. Comparative proteomic analysis using two-dimensional electrophoresis and mass spectrometry was performed to further investigate proteins and associated metabolic pathways imparting increased rhizome elongation by GA. A total of 37 and 38 differentially expressed proteins in response to GA3 treatment were identified in TF and KB plants, respectively, which were mainly involved in photosynthesis, energy and amino acid metabolism, protein synthesis, defense and cell development processes. Accelerated rhizome elongation in KB by GA could be mainly associated with the increased abundance of proteins involved in energy metabolism (glyceraldehyde-3-phosphate dehydrogenase, fructose-bisphosphate aldolase, and ATP synthase), amino acid metabolism (S-adenosylmethionine and adenosylhomocysteinase), protein synthesis (HSP90, elongation factor Tu and eukaryotic translation initiation factor 5A), cell-wall development (cell dividion cycle protein, alpha tubulin-2A and actin), and signal transduction (calreticulin). These proteins could be used as candidate proteins for further analysis of molecular mechanisms controlling rhizome growth. PMID:27446135

77 FR 39986 - Information Collection; Health Screening Questionnaire

Federal Register 2010, 2011, 2012, 2013, 2014

2012-07-06

... DEPARTMENT OF AGRICULTURE Forest Service Information Collection; Health Screening Questionnaire... organizations on the extension of a currently approved information collection, Health Screening Questionnaire... holidays. SUPPLEMENTARY INFORMATION: Title: Health Screening Questionnaire. OMB Number: 0596-0164...

Discovery Proteomics Identifies a Molecular Link between the Coatomer Protein Complex I and Androgen Receptor-dependent Transcription*

PubMed Central

Hsiao, Jordy J.; Smits, Melinda M.; Ng, Brandon H.; Lee, Jinhee; Wright, Michael E.

2016-01-01

Aberrant androgen receptor (AR)-dependent transcription is a hallmark of human prostate cancers. At the molecular level, ligand-mediated AR activation is coordinated through spatial and temporal protein-protein interactions involving AR-interacting proteins, which we designate the “AR-interactome.” Despite many years of research, the ligand-sensitive protein complexes involved in ligand-mediated AR activation in prostate tumor cells have not been clearly defined. Here, we describe the development, characterization, and utilization of a novel human LNCaP prostate tumor cell line, N-AR, which stably expresses wild-type AR tagged at its N terminus with the streptavidin-binding peptide epitope (streptavidin-binding peptide-tagged wild-type androgen receptor; SBP-AR). A bioanalytical workflow involving streptavidin chromatography and label-free quantitative mass spectrometry was used to identify SBP-AR and associated ligand-sensitive cytosolic proteins/protein complexes linked to AR activation in prostate tumor cells. Functional studies verified that ligand-sensitive proteins identified in the proteomic screen encoded modulators of AR-mediated transcription, suggesting that these novel proteins were putative SBP-AR-interacting proteins in N-AR cells. This was supported by biochemical associations between recombinant SBP-AR and the ligand-sensitive coatomer protein complex I (COPI) retrograde trafficking complex in vitro. Extensive biochemical and molecular experiments showed that the COPI retrograde complex regulates ligand-mediated AR transcriptional activation, which correlated with the mobilization of the Golgi-localized ARA160 coactivator to the nuclear compartment of prostate tumor cells. Collectively, this study provides a bioanalytical strategy to validate the AR-interactome and define novel AR-interacting proteins involved in ligand-mediated AR activation in prostate tumor cells. Moreover, we describe a cellular system to study how compartment-specific AR-interacting proteins influence AR activation and contribute to aberrant AR-dependent transcription that underlies the majority of human prostate cancers. PMID:27365400

MEGGASENSE - The Metagenome/Genome Annotated Sequence Natural Language Search Engine: A Platform for 
the Construction of Sequence Data Warehouses.

PubMed

Gacesa, Ranko; Zucko, Jurica; Petursdottir, Solveig K; Gudmundsdottir, Elisabet Eik; Fridjonsson, Olafur H; Diminic, Janko; Long, Paul F; Cullum, John; Hranueli, Daslav; Hreggvidsson, Gudmundur O; Starcevic, Antonio

2017-06-01

The MEGGASENSE platform constructs relational databases of DNA or protein sequences. The default functional analysis uses 14 106 hidden Markov model (HMM) profiles based on sequences in the KEGG database. The Solr search engine allows sophisticated queries and a BLAST search function is also incorporated. These standard capabilities were used to generate the SCATT database from the predicted proteome of Streptomyces cattleya . The implementation of a specialised metagenome database (AMYLOMICS) for bioprospecting of carbohydrate-modifying enzymes is described. In addition to standard assembly of reads, a novel 'functional' assembly was developed, in which screening of reads with the HMM profiles occurs before the assembly. The AMYLOMICS database incorporates additional HMM profiles for carbohydrate-modifying enzymes and it is illustrated how the combination of HMM and BLAST analyses helps identify interesting genes. A variety of different proteome and metagenome databases have been generated by MEGGASENSE.

RNA deep sequencing as a tool for selection of cell lines for systematic subcellular localization of all human proteins.

PubMed

Danielsson, Frida; Wiking, Mikaela; Mahdessian, Diana; Skogs, Marie; Ait Blal, Hammou; Hjelmare, Martin; Stadler, Charlotte; Uhlén, Mathias; Lundberg, Emma

2013-01-04

One of the major challenges of a chromosome-centric proteome project is to explore in a systematic manner the potential proteins identified from the chromosomal genome sequence, but not yet characterized on a protein level. Here, we describe the use of RNA deep sequencing to screen human cell lines for RNA profiles and to use this information to select cell lines suitable for characterization of the corresponding gene product. In this manner, the subcellular localization of proteins can be analyzed systematically using antibody-based confocal microscopy. We demonstrate the usefulness of selecting cell lines with high expression levels of RNA transcripts to increase the likelihood of high quality immunofluorescence staining and subsequent successful subcellular localization of the corresponding protein. The results show a path to combine transcriptomics with affinity proteomics to characterize the proteins in a gene- or chromosome-centric manner.

Modeling the Cost-Effectiveness of Alternative Upper Age Limits for Breast Cancer Screening in England and Wales.

PubMed

Rafia, Rachid; Brennan, Alan; Madan, Jason; Collins, Karen; Reed, Malcolm W R; Lawrence, Gill; Robinson, Thompson; Greenberg, David; Wyld, Lynda

2016-06-01

Currently in the United Kingdom, the National Health Service (NHS) Breast Screening Programme invites all women for triennial mammography between the ages of 47 and 73 years (the extension to 47-50 and 70-73 years is currently examined as part of a randomized controlled trial). The benefits and harms of screening in women 70 years and older, however, are less well documented. The aim of this study was to examine whether extending screening to women older than 70 years would represent a cost-effective use of NHS resources and to identify the upper age limit at which screening mammography should be extended in England and Wales. A mathematical model that allows the impact of screening policies on cancer diagnosis and subsequent management to be assessed was built. The model has two parts: a natural history model of the progression of breast cancer up to discovery and a postdiagnosis model of treatment, recurrence, and survival. The natural history model was calibrated to available data and compared against published literature. The management of breast cancer at diagnosis was taken from registry data and valued using official UK tariffs. The model estimated that screening would lead to overdiagnosis in 6.2% of screen-detected women at the age of 72 years, increasing up to 37.9% at the age of 90 years. Under commonly quoted willingness-to-pay thresholds in the United Kingdom, our study suggests that an extension to screening up to the age of 78 years represents a cost-effective strategy. This study provides encouraging findings to support the extension of the screening program to older ages and suggests that further extension of the UK NHS Breast Screening Programme up to age 78 years beyond the current upper age limit of 73 years could be potentially cost-effective according to current NHS willingness-to-pay thresholds. Copyright © 2016 International Society for Pharmacoeconomics and Outcomes Research (ISPOR). Published by Elsevier Inc. All rights reserved.

The Future of Weapons of Mass Destruction: Their Nature and Role in 2030

DTIC Science & Technology

2014-06-01

substantial improvements are al- lowed under the rubric of life extension. Other states are not so constrained and may find different ways to develop pure...The foregoing capabilities do not involve genetic manipulation or bioen- gineering; they utilize longstanding biological knowledge and processes. More...sophisticated understanding of biological systems (genomic and proteomic infor- mation) and processes ( genetic modification, bioengineering) for

Stereo 3D vision adapter using commercial DIY goods

NASA Astrophysics Data System (ADS)

Sakamoto, Kunio; Ohara, Takashi

2009-10-01

The conventional display can show only one screen, but it is impossible to enlarge the size of a screen, for example twice. Meanwhile the mirror supplies us with the same image but this mirror image is usually upside down. Assume that the images on an original screen and a virtual screen in the mirror are completely different and both images can be displayed independently. It would be possible to enlarge a screen area twice. This extension method enables the observers to show the virtual image plane and to enlarge a screen area twice. Although the displaying region is doubled, this virtual display could not produce 3D images. In this paper, we present an extension method using a unidirectional diffusing image screen and an improvement for displaying a 3D image using orthogonal polarized image projection.

Generalizing Screen Inferiority--Does the Medium, Screen versus Paper, Affect Performance Even with Brief Tasks?

ERIC Educational Resources Information Center

Sidi, Yael; Ophir, Yael; Ackerman, Rakefet

2016-01-01

Screen inferiority in performance and metacognitive processes has been repeatedly found with text learning. Common explanations for screen inferiority relate to technological and physiological disadvantages associated with extensive reading on screen. However, recent studies point to lesser recruitment of mental effort on screen than on paper.…

Proteomic Analysis of Pachytene Spermatocytes of Sterile Hybrid Male Mice.

PubMed

Wang, Lu; Guo, Yueshuai; Liu, Wenjing; Zhao, Weidong; Song, Gendi; Zhou, Tao; Huang, Hefeng; Guo, Xuejiang; Sun, Fei

2016-09-01

Incompatibilities in interspecific hybrids, such as reduced hybrid fertility and lethality, are common features resulting from reproductive isolation that lead to speciation. Subspecies crosses of house mice produce offspring in which one sex is infertile or absent, yet the molecular mechanisms of hybrid sterility are poorly understood. In this study, we observed extensive asynapsis of chromosomes and disturbance of the sex body in pachytene spermatocytes of sterile F1 males (PWK/Ph female × C57BL/6J male). We report the high-confidence identification of 4005 proteins in the pachytene spermatocytes of fertile F1 males (PWK/Ph male × C57BL/6J female) and sterile F1 males (PWK/Ph female × C57BL/6J male), of which 215 were upregulated and 381 were downregulated. Bioinformatics analysis of the proteome led to the identification of 43 and 59 proteins known to be essential for male meiosis and spermatogenesis in mice, respectively. Characterization of the proteome of pachytene spermatocytes associated with hybrid male sterility provides an inventory of proteins that is useful for understanding meiosis and the mechanisms of hybrid male infertility. © 2016 by the Society for the Study of Reproduction, Inc.

Environmental Interactions and Epistasis Are Revealed in the Proteomic Responses to Complex Stimuli

PubMed Central

Samir, Parimal; Rahul; Slaughter, James C.; Link, Andrew J.

2015-01-01

Ultimately, the genotype of a cell and its interaction with the environment determine the cell’s biochemical state. While the cell’s response to a single stimulus has been studied extensively, a conceptual framework to model the effect of multiple environmental stimuli applied concurrently is not as well developed. In this study, we developed the concepts of environmental interactions and epistasis to explain the responses of the S. cerevisiae proteome to simultaneous environmental stimuli. We hypothesize that, as an abstraction, environmental stimuli can be treated as analogous to genetic elements. This would allow modeling of the effects of multiple stimuli using the concepts and tools developed for studying gene interactions. Mirroring gene interactions, our results show that environmental interactions play a critical role in determining the state of the proteome. We show that individual and complex environmental stimuli behave similarly to genetic elements in regulating the cellular responses to stimuli, including the phenomena of dominance and suppression. Interestingly, we observed that the effect of a stimulus on a protein is dominant over other stimuli if the response to the stimulus involves the protein. Using publicly available transcriptomic data, we find that environmental interactions and epistasis regulate transcriptomic responses as well. PMID:26247773

Evaluation of formalin-fixed paraffin-embedded tissues in the proteomic analysis of parathyroid glands

PubMed Central

2011-01-01

Background Proteomic research in the field of parathyroid tissues is limited by the very small dimension of the glands and by the low incidence of cancer lesions (1%). Formalin-fixed paraffin-embedded (FFPE) tissue specimens are a potentially valuable resource for discovering protein cancer biomarkers. In this study we have verified the applicability of a heat induced protein extraction from FFPE parathyroid adenoma tissues followed by a gel-based or gel-free proteomic approach in order to achieve protein separation and identification. Results The best results for high quality MS spectra and parameters, were obtained by using a gel-free approach, and up to 163 unique proteins were identified. Similar results were obtained by applying both SDS-out and SDS-out + TCA/Acetone techniques during the gel-free method. Western blot analysis carried out with specific antibodies suggested that the antigenicity was not always preserved, while specific immunoreactions were detected for calmodulin, B box and SPRY domain-containing protein (BSPRY), peroxiredoxin 6 (PRDX 6) and parvalbumin. Conclusions In spite of some limitations mainly due to the extensive formalin-induced covalent cross-linking, our results essentially suggest the applicability of a proteomic approach to FFPE parathyroid specimens. From our point of view, FFPE extracts might be an alternative source, especially in the validation phase of protein biomarkers when a large cohort of samples is required and the low availability of frozen tissues might be constraining. PMID:21651755

41 CFR 102-75.1265 - Are extensions granted to the Federal screening and response timeframes?

Code of Federal Regulations, 2010 CFR

2010-07-01

... and Property Management Federal Property Management Regulations System (Continued) FEDERAL MANAGEMENT REGULATION REAL PROPERTY 75-REAL PROPERTY DISPOSAL Screening of Federal Real Property § 102-75.1265 Are... request an extension of time to submit their formal transfer request if they are not promptly provided GSA...

Cellulose and hemicellulose decomposition by forest soil bacteria proceeds by the action of structurally variable enzymatic systems

PubMed Central

López-Mondéjar, Rubén; Zühlke, Daniela; Becher, Dörte; Riedel, Katharina; Baldrian, Petr

2016-01-01

Evidence shows that bacteria contribute actively to the decomposition of cellulose and hemicellulose in forest soil; however, their role in this process is still unclear. Here we performed the screening and identification of bacteria showing potential cellulolytic activity from litter and organic soil of a temperate oak forest. The genomes of three cellulolytic isolates previously described as abundant in this ecosystem were sequenced and their proteomes were characterized during the growth on plant biomass and on microcrystalline cellulose. Pedobacter and Mucilaginibacter showed complex enzymatic systems containing highly diverse carbohydrate-active enzymes for the degradation of cellulose and hemicellulose, which were functionally redundant for endoglucanases, β-glucosidases, endoxylanases, β-xylosidases, mannosidases and carbohydrate-binding modules. Luteibacter did not express any glycosyl hydrolases traditionally recognized as cellulases. Instead, cellulose decomposition was likely performed by an expressed GH23 family protein containing a cellulose-binding domain. Interestingly, the presence of plant lignocellulose as well as crystalline cellulose both trigger the production of a wide set of hydrolytic proteins including cellulases, hemicellulases and other glycosyl hydrolases. Our findings highlight the extensive and unexplored structural diversity of enzymatic systems in cellulolytic soil bacteria and indicate the roles of multiple abundant bacterial taxa in the decomposition of cellulose and other plant polysaccharides. PMID:27125755

Identification of candidate angiogenic inhibitors processed by matrix metalloproteinase 2 (MMP-2) in cell-based proteomic screens: disruption of vascular endothelial growth factor (VEGF)/heparin affin regulatory peptide (pleiotrophin) and VEGF/Connective tissue growth factor angiogenic inhibitory complexes by MMP-2 proteolysis.

PubMed

Dean, Richard A; Butler, Georgina S; Hamma-Kourbali, Yamina; Delbé, Jean; Brigstock, David R; Courty, José; Overall, Christopher M

2007-12-01

Matrix metalloproteinases (MMPs) exert both pro- and antiangiogenic functions by the release of cytokines or proteolytically generated angiogenic inhibitors from extracellular matrix and basement membrane remodeling. In the Mmp2-/- mouse neovascularization is greatly reduced, but the mechanistic aspects of this remain unclear. Using isotope-coded affinity tag labeling of proteins analyzed by multidimensional liquid chromatography and tandem mass spectrometry we explored proteome differences between Mmp2-/- cells and those rescued by MMP-2 transfection. Proteome signatures that are hallmarks of proteolysis revealed cleavage of many known MMP-2 substrates in the cellular context. Proteomic evidence of MMP-2 processing of novel substrates was found. Insulin-like growth factor binding protein 6, follistatin-like 1, and cystatin C protein cleavage by MMP-2 was biochemically confirmed, and the cleavage sites in heparin affin regulatory peptide (HARP; pleiotrophin) and connective tissue growth factor (CTGF) were sequenced by matrix-assisted laser desorption ionization-time of flight mass spectrometry. MMP-2 processing of HARP and CTGF released vascular endothelial growth factor (VEGF) from angiogenic inhibitory complexes. The cleaved HARP N-terminal domain increased HARP-induced cell proliferation, whereas the HARP C-terminal domain was antagonistic and decreased cell proliferation and migration. Hence the unmasking of cytokines, such as VEGF, by metalloproteinase processing of their binding proteins is a new mechanism in the control of cytokine activation and angiogenesis.

Quantitative proteomic analysis of bacterial enzymes released in cheese during ripening.

PubMed

Jardin, Julien; Mollé, Daniel; Piot, Michel; Lortal, Sylvie; Gagnaire, Valérie

2012-04-02

Due to increasingly available bacterial genomes in databases, proteomic tools have recently been used to screen proteins expressed by micro-organisms in food in order to better understand their metabolism in situ. While the main objective is the systematic identification of proteins, the next step will be to bridge the gap between identification and quantification of these proteins. For that purpose, a new mass spectrometry-based approach was applied, using isobaric tagging reagent for quantitative proteomic analysis (iTRAQ), which are amine specific and yield labelled peptides identical in mass. Experimental Swiss-type cheeses were manufactured from microfiltered milk using Streptococcus thermophilus ITG ST20 and Lactobacillus helveticus ITG LH1 as lactic acid starters. At three ripening times (7, 20 and 69 days), cheese aqueous phases were extracted and enriched in bacterial proteins by fractionation. Each sample, standardised in protein amount prior to proteomic analyses, was: i) analysed by 2D-electrophoresis for qualitative analysis and ii) submitted to trypsinolysis, and labelled with specific iTRAQ tag, one per ripening time. The three labelled samples were mixed together and analysed by nano-LC coupled on-line with ESI-QTOF mass spectrometer. Thirty proteins, both from bacterial or bovine origin, were identified and efficiently quantified. The free bacterial proteins detected were enzymes from the central carbon metabolism as well as stress proteins. Depending on the protein considered, the quantity of these proteins in the cheese aqueous extract increased from 2.5 to 20 fold in concentration from day 7 to day 69 of ripening. Copyright © 2012 Elsevier B.V. All rights reserved.

Respiratory Mucosal Proteome Quantification in Human Influenza Infections.

PubMed

Marion, Tony; Elbahesh, Husni; Thomas, Paul G; DeVincenzo, John P; Webby, Richard; Schughart, Klaus

2016-01-01

Respiratory influenza virus infections represent a serious threat to human health. Underlying medical conditions and genetic make-up predispose some influenza patients to more severe forms of disease. To date, only a few studies have been performed in patients to correlate a selected group of cytokines and chemokines with influenza infection. Therefore, we evaluated the potential of a novel multiplex micro-proteomics technology, SOMAscan, to quantify proteins in the respiratory mucosa of influenza A and B infected individuals. The analysis included but was not limited to quantification of cytokines and chemokines detected in previous studies. SOMAscan quantified more than 1,000 secreted proteins in small nasal wash volumes from infected and healthy individuals. Our results illustrate the utility of micro-proteomic technology for analysis of proteins in small volumes of respiratory mucosal samples. Furthermore, when we compared nasal wash samples from influenza-infected patients with viral load ≥ 2(8) and increased IL-6 and CXCL10 to healthy controls, we identified 162 differentially-expressed proteins between the two groups. This number greatly exceeds the number of DEPs identified in previous studies in human influenza patients. Most of the identified proteins were associated with the host immune response to infection, and changes in protein levels of 151 of the DEPs were significantly correlated with viral load. Most important, SOMAscan identified differentially expressed proteins heretofore not associated with respiratory influenza infection in humans. Our study is the first report for the use of SOMAscan to screen nasal secretions. It establishes a precedent for micro-proteomic quantification of proteins that reflect ongoing response to respiratory infection.

Respiratory Mucosal Proteome Quantification in Human Influenza Infections

PubMed Central

Marion, Tony; Elbahesh, Husni; Thomas, Paul G.; DeVincenzo, John P.; Webby, Richard; Schughart, Klaus

2016-01-01

Respiratory influenza virus infections represent a serious threat to human health. Underlying medical conditions and genetic make-up predispose some influenza patients to more severe forms of disease. To date, only a few studies have been performed in patients to correlate a selected group of cytokines and chemokines with influenza infection. Therefore, we evaluated the potential of a novel multiplex micro-proteomics technology, SOMAscan, to quantify proteins in the respiratory mucosa of influenza A and B infected individuals. The analysis included but was not limited to quantification of cytokines and chemokines detected in previous studies. SOMAscan quantified more than 1,000 secreted proteins in small nasal wash volumes from infected and healthy individuals. Our results illustrate the utility of micro-proteomic technology for analysis of proteins in small volumes of respiratory mucosal samples. Furthermore, when we compared nasal wash samples from influenza-infected patients with viral load ≥ 28 and increased IL-6 and CXCL10 to healthy controls, we identified 162 differentially-expressed proteins between the two groups. This number greatly exceeds the number of DEPs identified in previous studies in human influenza patients. Most of the identified proteins were associated with the host immune response to infection, and changes in protein levels of 151 of the DEPs were significantly correlated with viral load. Most important, SOMAscan identified differentially expressed proteins heretofore not associated with respiratory influenza infection in humans. Our study is the first report for the use of SOMAscan to screen nasal secretions. It establishes a precedent for micro-proteomic quantification of proteins that reflect ongoing response to respiratory infection. PMID:27088501

Identification of Candidate Angiogenic Inhibitors Processed by Matrix Metalloproteinase 2 (MMP-2) in Cell-Based Proteomic Screens: Disruption of Vascular Endothelial Growth Factor (VEGF)/Heparin Affin Regulatory Peptide (Pleiotrophin) and VEGF/Connective Tissue Growth Factor Angiogenic Inhibitory Complexes by MMP-2 Proteolysis▿ †

PubMed Central

Dean, Richard A.; Butler, Georgina S.; Hamma-Kourbali, Yamina; Delbé, Jean; Brigstock, David R.; Courty, José; Overall, Christopher M.

2007-01-01

Matrix metalloproteinases (MMPs) exert both pro- and antiangiogenic functions by the release of cytokines or proteolytically generated angiogenic inhibitors from extracellular matrix and basement membrane remodeling. In the Mmp2−/− mouse neovascularization is greatly reduced, but the mechanistic aspects of this remain unclear. Using isotope-coded affinity tag labeling of proteins analyzed by multidimensional liquid chromatography and tandem mass spectrometry we explored proteome differences between Mmp2−/− cells and those rescued by MMP-2 transfection. Proteome signatures that are hallmarks of proteolysis revealed cleavage of many known MMP-2 substrates in the cellular context. Proteomic evidence of MMP-2 processing of novel substrates was found. Insulin-like growth factor binding protein 6, follistatin-like 1, and cystatin C protein cleavage by MMP-2 was biochemically confirmed, and the cleavage sites in heparin affin regulatory peptide (HARP; pleiotrophin) and connective tissue growth factor (CTGF) were sequenced by matrix-assisted laser desorption ionization-time of flight mass spectrometry. MMP-2 processing of HARP and CTGF released vascular endothelial growth factor (VEGF) from angiogenic inhibitory complexes. The cleaved HARP N-terminal domain increased HARP-induced cell proliferation, whereas the HARP C-terminal domain was antagonistic and decreased cell proliferation and migration. Hence the unmasking of cytokines, such as VEGF, by metalloproteinase processing of their binding proteins is a new mechanism in the control of cytokine activation and angiogenesis. PMID:17908800

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

PubMed Central

Haraszti, Reka A.; Didiot, Marie-Cecile; Sapp, Ellen; Leszyk, John; Shaffer, Scott A.; Rockwell, Hannah E.; Gao, Fei; Narain, Niven R.; DiFiglia, Marian; Kiebish, Michael A.; Aronin, Neil; Khvorova, Anastasia

2016-01-01

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. PMID:27863537

A Multilayer Network Approach for Guiding Drug Repositioning in Neglected Diseases

PubMed Central

Chernomoretz, Ariel; Agüero, Fernán

2016-01-01

Drug development for neglected diseases has been historically hampered due to lack of market incentives. The advent of public domain resources containing chemical information from high throughput screenings is changing the landscape of drug discovery for these diseases. In this work we took advantage of data from extensively studied organisms like human, mouse, E. coli and yeast, among others, to develop a novel integrative network model to prioritize and identify candidate drug targets in neglected pathogen proteomes, and bioactive drug-like molecules. We modeled genomic (proteins) and chemical (bioactive compounds) data as a multilayer weighted network graph that takes advantage of bioactivity data across 221 species, chemical similarities between 1.7 105 compounds and several functional relations among 1.67 105 proteins. These relations comprised orthology, sharing of protein domains, and shared participation in defined biochemical pathways. We showcase the application of this network graph to the problem of prioritization of new candidate targets, based on the information available in the graph for known compound-target associations. We validated this strategy by performing a cross validation procedure for known mouse and Trypanosoma cruzi targets and showed that our approach outperforms classic alignment-based approaches. Moreover, our model provides additional flexibility as two different network definitions could be considered, finding in both cases qualitatively different but sensible candidate targets. We also showcase the application of the network to suggest targets for orphan compounds that are active against Plasmodium falciparum in high-throughput screens. In this case our approach provided a reduced prioritization list of target proteins for the query molecules and showed the ability to propose new testable hypotheses for each compound. Moreover, we found that some predictions highlighted by our network model were supported by independent experimental validations as found post-facto in the literature. PMID:26735851

A Multilayer Network Approach for Guiding Drug Repositioning in Neglected Diseases.

PubMed

Berenstein, Ariel José; Magariños, María Paula; Chernomoretz, Ariel; Agüero, Fernán

2016-01-01

Drug development for neglected diseases has been historically hampered due to lack of market incentives. The advent of public domain resources containing chemical information from high throughput screenings is changing the landscape of drug discovery for these diseases. In this work we took advantage of data from extensively studied organisms like human, mouse, E. coli and yeast, among others, to develop a novel integrative network model to prioritize and identify candidate drug targets in neglected pathogen proteomes, and bioactive drug-like molecules. We modeled genomic (proteins) and chemical (bioactive compounds) data as a multilayer weighted network graph that takes advantage of bioactivity data across 221 species, chemical similarities between 1.7 105 compounds and several functional relations among 1.67 105 proteins. These relations comprised orthology, sharing of protein domains, and shared participation in defined biochemical pathways. We showcase the application of this network graph to the problem of prioritization of new candidate targets, based on the information available in the graph for known compound-target associations. We validated this strategy by performing a cross validation procedure for known mouse and Trypanosoma cruzi targets and showed that our approach outperforms classic alignment-based approaches. Moreover, our model provides additional flexibility as two different network definitions could be considered, finding in both cases qualitatively different but sensible candidate targets. We also showcase the application of the network to suggest targets for orphan compounds that are active against Plasmodium falciparum in high-throughput screens. In this case our approach provided a reduced prioritization list of target proteins for the query molecules and showed the ability to propose new testable hypotheses for each compound. Moreover, we found that some predictions highlighted by our network model were supported by independent experimental validations as found post-facto in the literature.

Comprehensive prediction of drug-protein interactions and side effects for the human proteome

PubMed Central

Zhou, Hongyi; Gao, Mu; Skolnick, Jeffrey

2015-01-01

Identifying unexpected drug-protein interactions is crucial for drug repurposing. We develop a comprehensive proteome scale approach that predicts human protein targets and side effects of drugs. For drug-protein interaction prediction, FINDSITEcomb, whose average precision is ~30% and recall ~27%, is employed. For side effect prediction, a new method is developed with a precision of ~57% and a recall of ~24%. Our predictions show that drugs are quite promiscuous, with the average (median) number of human targets per drug of 329 (38), while a given protein interacts with 57 drugs. The result implies that drug side effects are inevitable and existing drugs may be useful for repurposing, with only ~1,000 human proteins likely causing serious side effects. A killing index derived from serious side effects has a strong correlation with FDA approved drugs being withdrawn. Therefore, it provides a pre-filter for new drug development. The methodology is free to the academic community on the DR. PRODIS (DRugome, PROteome, and DISeasome) webserver at http://cssb.biology.gatech.edu/dr.prodis/. DR. PRODIS provides protein targets of drugs, drugs for a given protein target, associated diseases and side effects of drugs, as well as an interface for the virtual target screening of new compounds. PMID:26057345

PeroxisomeDB: a database for the peroxisomal proteome, functional genomics and disease

PubMed Central

Schlüter, Agatha; Fourcade, Stéphane; Domènech-Estévez, Enric; Gabaldón, Toni; Huerta-Cepas, Jaime; Berthommier, Guillaume; Ripp, Raymond; Wanders, Ronald J. A.; Poch, Olivier; Pujol, Aurora

2007-01-01

Peroxisomes are essential organelles of eukaryotic origin, ubiquitously distributed in cells and organisms, playing key roles in lipid and antioxidant metabolism. Loss or malfunction of peroxisomes causes more than 20 fatal inherited conditions. We have created a peroxisomal database () that includes the complete peroxisomal proteome of Homo sapiens and Saccharomyces cerevisiae, by gathering, updating and integrating the available genetic and functional information on peroxisomal genes. PeroxisomeDB is structured in interrelated sections ‘Genes’, ‘Functions’, ‘Metabolic pathways’ and ‘Diseases’, that include hyperlinks to selected features of NCBI, ENSEMBL and UCSC databases. We have designed graphical depictions of the main peroxisomal metabolic routes and have included updated flow charts for diagnosis. Precomputed BLAST, PSI-BLAST, multiple sequence alignment (MUSCLE) and phylogenetic trees are provided to assist in direct multispecies comparison to study evolutionary conserved functions and pathways. Highlights of the PeroxisomeDB include new tools developed for facilitating (i) identification of novel peroxisomal proteins, by means of identifying proteins carrying peroxisome targeting signal (PTS) motifs, (ii) detection of peroxisomes in silico, particularly useful for screening the deluge of newly sequenced genomes. PeroxisomeDB should contribute to the systematic characterization of the peroxisomal proteome and facilitate system biology approaches on the organelle. PMID:17135190

Proteomic characterization of fresh spermatozoa and supernatant after cryopreservation in relation to freezability of carp (Cyprinus carpio L) semen

PubMed Central

Ciereszko, Andrzej

2018-01-01

Our recent studies suggested that the freezability of carp semen is related to seminal plasma protein profiles. Here, we aimed to compare the spermatozoa proteomes of good (GF) and poor (PF) freezability semen of carp. To achieve this, we used two-dimensional difference in gel electrophoresis followed by MALDI-TOF/TOF mass spectrometry. The semen was classified as GF or PF based on sperm motility after freeze/thawing. We identified proteins enriched in spermatozoa of GF (22 proteins) and PF (18 proteins) semen. We also identified 12 proteins enriched in the supernatant after cryopreservation of PF semen. Good freezability is related to high concentrations of proteins involved in the maintenance of flagella structure, membrane fluidity, efficient control of Ca2+ and sperm motility, energy production, and antioxidative protection, which likely reflects the full maturation status of spermatozoa of GF semen. On the other hand poor freezability seems to be related to the presence of proteins identified as released in high quantities from cryopreserved sperm of PF. Thus, the identified proteins might be useful bioindicators of freezing resilience and could be used to screen carp males before cryopreservation, thus improve long-term sperm preservation in carp. Data are available via ProteomeXchange with identifier PXD008187. PMID:29565997

Proteomic Analysis Reveals the Contribution of TGFβ/Smad4 Signaling Pathway to Cell Differentiation During Planarian Tail Regeneration.

PubMed

Chen, Xiaoguang; Xu, Cunshuan

2017-06-01

After planarian tail is cut off, posterior end of the remaining fragment will regenerate a new tail within about 1 week. However, many details of this process remain unclear up to date. For this reason, we performed the dynamic proteomic analysis of the regenerating tail fragments at 6, 12, 24, 72, 120, and 168 h post-amputation (hpa). Using two-dimensional electrophoresis (2-DE) in combination with MALDI-TOF-TOF/MS analysis, a total of 1088 peptides were identified as significantly changed between tail-cutting groups and 0-h group, 482 of which have identifiable protein names. Of these 482 proteins, there were 111 originating from the Turbellaria. Protein functional categorization showed that these 111 proteins are mainly related to differentiation and development, transcription and translation, cell signal transduction, and cell proliferation. The screening of key protein considered the transcription factor Smad4 as important protein for planarian tail regeneration. Cell signaling pathway analysis, combined with proteomic profiling of regenerating tail fragment, showed that TGFβ/Smad4 pathway was activated during planarian tail regeneration. Based on a comprehensive analysis of 2-DE MALDI-TOF-TOF/MS and bioinformatics analyses, it could be concluded that TGFβ/Smad4 pathway perhaps plays an important role in tail regeneration via promoting cell differentiation.

Functional Proteomics to Identify Moderators of CD8+ T-Cell Function in Melanoma

DTIC Science & Technology

2013-09-01

could then be used to develop imaging agents that are targeted to the TILs. We used an M13 phage vector, which displays a pentavalent peptide as...of the identified transcript. To directly indentify inhibitory molecules, we have proposed to use phage - display expression libraries to perform...of TCD8. 3. To determine the ligands for molecules that can modulate the TCD8 functional state. Body: A. Phage Screen Summary Phage display

Proteome-wide covalent ligand discovery in native biological systems

PubMed Central

Backus, Keriann M.; Correia, Bruno E.; Lum, Kenneth M.; Forli, Stefano; Horning, Benjamin D.; González-Páez, Gonzalo E.; Chatterjee, Sandip; Lanning, Bryan R.; Teijaro, John R.; Olson, Arthur J.; Wolan, Dennis W.; Cravatt, Benjamin F.

2016-01-01

Small molecules are powerful tools for investigating protein function and can serve as leads for new therapeutics. Most human proteins, however, lack small-molecule ligands, and entire protein classes are considered “undruggable” 1,2. Fragment-based ligand discovery (FBLD) can identify small-molecule probes for proteins that have proven difficult to target using high-throughput screening of complex compound libraries 1,3. Although reversibly binding ligands are commonly pursued, covalent fragments provide an alternative route to small-molecule probes 4–10, including those that can access regions of proteins that are difficult to access through binding affinity alone 5,10,11. In this manuscript, we report a quantitative analysis of cysteine-reactive small-molecule fragments screened against thousands of proteins. Covalent ligands were identified for >700 cysteines found in both druggable proteins and proteins deficient in chemical probes, including transcription factors, adaptor/scaffolding proteins, and uncharacterized proteins. Among the atypical ligand-protein interactions discovered were compounds that react preferentially with pro- (inactive) caspases. We used these ligands to distinguish extrinsic apoptosis pathways in human cell lines versus primary human T-cells, showing that the former is largely mediated by caspase-8 while the latter depends on both caspase-8 and −10. Fragment-based covalent ligand discovery provides a greatly expanded portrait of the ligandable proteome and furnishes compounds that can illuminate protein functions in native biological systems. PMID:27309814

A systems wide mass spectrometric based linear motif screen to identify dominant in-vivo interacting proteins for the ubiquitin ligase MDM2.

PubMed

Nicholson, Judith; Scherl, Alex; Way, Luke; Blackburn, Elizabeth A; Walkinshaw, Malcolm D; Ball, Kathryn L; Hupp, Ted R

2014-06-01

Linear motifs mediate protein-protein interactions (PPI) that allow expansion of a target protein interactome at a systems level. This study uses a proteomics approach and linear motif sub-stratifications to expand on PPIs of MDM2. MDM2 is a multi-functional protein with over one hundred known binding partners not stratified by hierarchy or function. A new linear motif based on a MDM2 interaction consensus is used to select novel MDM2 interactors based on Nutlin-3 responsiveness in a cell-based proteomics screen. MDM2 binds a subset of peptide motifs corresponding to real proteins with a range of allosteric responses to MDM2 ligands. We validate cyclophilin B as a novel protein with a consensus MDM2 binding motif that is stabilised by Nutlin-3 in vivo, thus identifying one of the few known interactors of MDM2 that is stabilised by Nutlin-3. These data invoke two modes of peptide binding at the MDM2 N-terminus that rely on a consensus core motif to control the equilibrium between MDM2 binding proteins. This approach stratifies MDM2 interacting proteins based on the linear motif feature and provides a new biomarker assay to define clinically relevant Nutlin-3 responsive MDM2 interactors. Copyright © 2014 Elsevier Inc. All rights reserved.

Proteomic Screening and Lasso Regression Reveal Differential Signaling in Insulin and Insulin-like Growth Factor I (IGF1) Pathways *

PubMed Central

Erdem, Cemal; Nagle, Alison M.; Casa, Angelo J.; Litzenburger, Beate C.; Wang, Yu-fen; Taylor, D. Lansing; Lee, Adrian V.; Lezon, Timothy R.

2016-01-01

Insulin and insulin-like growth factor I (IGF1) influence cancer risk and progression through poorly understood mechanisms. To better understand the roles of insulin and IGF1 signaling in breast cancer, we combined proteomic screening with computational network inference to uncover differences in IGF1 and insulin induced signaling. Using reverse phase protein array, we measured the levels of 134 proteins in 21 breast cancer cell lines stimulated with IGF1 or insulin for up to 48 h. We then constructed directed protein expression networks using three separate methods: (i) lasso regression, (ii) conventional matrix inversion, and (iii) entropy maximization. These networks, named here as the time translation models, were analyzed and the inferred interactions were ranked by differential magnitude to identify pathway differences. The two top candidates, chosen for experimental validation, were shown to regulate IGF1/insulin induced phosphorylation events. First, acetyl-CoA carboxylase (ACC) knock-down was shown to increase the level of mitogen-activated protein kinase (MAPK) phosphorylation. Second, stable knock-down of E-Cadherin increased the phospho-Akt protein levels. Both of the knock-down perturbations incurred phosphorylation responses stronger in IGF1 stimulated cells compared with insulin. Overall, the time-translation modeling coupled to wet-lab experiments has proven to be powerful in inferring differential interactions downstream of IGF1 and insulin signaling, in vitro. PMID:27364358

Screening of multiple myeloma by polyclonal rabbit anti-human plasmacytoma cell immunoglobulin.

PubMed

Mu, Bo; Zhang, Huan; Cai, Xiaoming; Yang, Junbao; Shen, Yuewu; Chen, Baofeng; Liang, Suhua

2013-01-01

Antibody-based immunotherapy has been effectively used for tumor treatment. However, to date, only a few tumor-associated antigens (TAAs) or therapeutic targets have been identified. Identification of more immunogenic antigens is essential for improvements in multiple myeloma (MM) diagnosis and therapy. In this study, we synthesized a polyclonal antibody (PAb) by immunizing rabbits with whole human plasmacytoma ARH-77 cells and identified MM-associated antigens, including enlonase, adipophilin, and HSP90s, among others, via proteomic technologies. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that 200 µg/mL PAb inhibits the proliferation of ARH-77 cells by over 50% within 48 h. Flow cytometric assay indicated that PAb treatment significantly increases the number of apoptotic cells compared with other treatments (52.1% vs. NS, 7.3% or control rabbit IgG, 9.9%). In vivo, PAb delayed tumor growth and prolonged the lifespan of mice. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that PAb also induces statistically significant changes in apoptosis compared with other treatments (P<0.05). We therefore conclude that PAb could be used for the effective screening and identification of TAA. PAb may have certain anti-tumor functions in vitro and in vivo. As such, its combination with proteomic technologies could be a promising approach for sieving TAA for the diagnosis and therapy of MM.

Quantitative proteomic analysis for high-throughput screening of differential glycoproteins in hepatocellular carcinoma serum

PubMed Central

Gao, Hua-Jun; Chen, Ya-Jing; Zuo, Duo; Xiao, Ming-Ming; Li, Ying; Guo, Hua; Zhang, Ning; Chen, Rui-Bing

2015-01-01

Objective Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths. Novel serum biomarkers are required to increase the sensitivity and specificity of serum screening for early HCC diagnosis. This study employed a quantitative proteomic strategy to analyze the differential expression of serum glycoproteins between HCC and normal control serum samples. Methods Lectin affinity chromatography (LAC) was used to enrich glycoproteins from the serum samples. Quantitative mass spectrometric analysis combined with stable isotope dimethyl labeling and 2D liquid chromatography (LC) separations were performed to examine the differential levels of the detected proteins between HCC and control serum samples. Western blot was used to analyze the differential expression levels of the three serum proteins. Results A total of 2,280 protein groups were identified in the serum samples from HCC patients by using the 2D LC-MS/MS method. Up to 36 proteins were up-regulated in the HCC serum, whereas 19 proteins were down-regulated. Three differential glycoproteins, namely, fibrinogen gamma chain (FGG), FOS-like antigen 2 (FOSL2), and α-1,6-mannosylglycoprotein 6-β-N-acetylglucosaminyltransferase B (MGAT5B) were validated by Western blot. All these three proteins were up-regulated in the HCC serum samples. Conclusion A quantitative glycoproteomic method was established and proven useful to determine potential novel biomarkers for HCC. PMID:26487969

Extensive Antibody Cross-reactivity among Infectious Gram-negative Bacteria Revealed by Proteome Microarray Analysis

DTIC Science & Technology

2009-08-27

aeruginosa, Salmonella ty- phimurium, Shigella flexneri, and Escherichia coli, all pathogenic Gram-negative bacteria. These antibodies en- abled detection of...w .m cponline.org D ow nloaded from broad range of O and H strains), Shigella flexneri (recognizing Shigella dysenteriae, Shigella boydii, and S...treated with antibiotics. In addition, sera were also obtained from rhesus monkeys vaccinated with the anthrax vaccine B. anthracis recombinant

Photosensitized UVA-Induced Cross-Linking between Human DNA Repair and Replication Proteins and DNA Revealed by Proteomic Analysis

PubMed Central

2016-01-01

Long wavelength ultraviolet radiation (UVA, 320–400 nm) interacts with chromophores present in human cells to induce reactive oxygen species (ROS) that damage both DNA and proteins. ROS levels are amplified, and the damaging effects of UVA are exacerbated if the cells are irradiated in the presence of UVA photosensitizers such as 6-thioguanine (6-TG), a strong UVA chromophore that is extensively incorporated into the DNA of dividing cells, or the fluoroquinolone antibiotic ciprofloxacin. Both DNA-embedded 6-TG and ciprofloxacin combine synergistically with UVA to generate high levels of ROS. Importantly, the extensive protein damage induced by these photosensitizer+UVA combinations inhibits DNA repair. DNA is maintained in intimate contact with the proteins that effect its replication, transcription, and repair, and DNA–protein cross-links (DPCs) are a recognized reaction product of ROS. Cross-linking of DNA metabolizing proteins would compromise these processes by introducing physical blocks and by depleting active proteins. We describe a sensitive and statistically rigorous method to analyze DPCs in cultured human cells. Application of this proteomics-based analysis to cells treated with 6-TG+UVA and ciprofloxacin+UVA identified proteins involved in DNA repair, replication, and gene expression among those most vulnerable to cross-linking under oxidative conditions. PMID:27654267

ATAQS: A computational software tool for high throughput transition optimization and validation for selected reaction monitoring mass spectrometry

PubMed Central

2011-01-01

Background Since its inception, proteomics has essentially operated in a discovery mode with the goal of identifying and quantifying the maximal number of proteins in a sample. Increasingly, proteomic measurements are also supporting hypothesis-driven studies, in which a predetermined set of proteins is consistently detected and quantified in multiple samples. Selected reaction monitoring (SRM) is a targeted mass spectrometric technique that supports the detection and quantification of specific proteins in complex samples at high sensitivity and reproducibility. Here, we describe ATAQS, an integrated software platform that supports all stages of targeted, SRM-based proteomics experiments including target selection, transition optimization and post acquisition data analysis. This software will significantly facilitate the use of targeted proteomic techniques and contribute to the generation of highly sensitive, reproducible and complete datasets that are particularly critical for the discovery and validation of targets in hypothesis-driven studies in systems biology. Result We introduce a new open source software pipeline, ATAQS (Automated and Targeted Analysis with Quantitative SRM), which consists of a number of modules that collectively support the SRM assay development workflow for targeted proteomic experiments (project management and generation of protein, peptide and transitions and the validation of peptide detection by SRM). ATAQS provides a flexible pipeline for end-users by allowing the workflow to start or end at any point of the pipeline, and for computational biologists, by enabling the easy extension of java algorithm classes for their own algorithm plug-in or connection via an external web site. This integrated system supports all steps in a SRM-based experiment and provides a user-friendly GUI that can be run by any operating system that allows the installation of the Mozilla Firefox web browser. Conclusions Targeted proteomics via SRM is a powerful new technique that enables the reproducible and accurate identification and quantification of sets of proteins of interest. ATAQS is the first open-source software that supports all steps of the targeted proteomics workflow. ATAQS also provides software API (Application Program Interface) documentation that enables the addition of new algorithms to each of the workflow steps. The software, installation guide and sample dataset can be found in http://tools.proteomecenter.org/ATAQS/ATAQS.html PMID:21414234

Proteome changes in platelets after pathogen inactivation--an interlaboratory consensus.

PubMed

Prudent, Michel; D'Alessandro, Angelo; Cazenave, Jean-Pierre; Devine, Dana V; Gachet, Christian; Greinacher, Andreas; Lion, Niels; Schubert, Peter; Steil, Leif; Thiele, Thomas; Tissot, Jean-Daniel; Völker, Uwe; Zolla, Lello

2014-04-01

Pathogen inactivation (PI) of platelet concentrates (PCs) reduces the proliferation/replication of a large range of bacteria, viruses, and parasites as well as residual leucocytes. Pathogen-inactivated PCs were evaluated in various clinical trials showing their efficacy and safety. Today, there is some debate over the hemostatic activity of treated PCs as the overall survival of PI platelets seems to be somewhat reduced, and in vitro measurements have identified some alterations in platelet function. Although the specific lesions resulting from PI of PCs are still not fully understood, proteomic studies have revealed potential damages at the protein level. This review merges the key findings of the proteomic analyses of PCs treated by the Mirasol Pathogen Reduction Technology, the Intercept Blood System, and the Theraflex UV-C system, respectively, and discusses the potential impact on the biological functions of platelets. The complementarities of the applied proteomic approaches allow the coverage of a wide range of proteins and provide a comprehensive overview of PI-mediated protein damage. It emerges that there is a relatively weak impact of PI on the overall proteome of platelets. However, some data show that the different PI treatments lead to an acceleration of platelet storage lesions, which is in agreement with the current model of platelet storage lesion in pathogen-inactivated PCs. Overall, the impact of the PI treatment on the proteome appears to be different among the PI systems. Mirasol impacts adhesion and platelet shape change, whereas Intercept seems to impact proteins of intracellular platelet activation pathways. Theraflex influences platelet shape change and aggregation, but the data reported to date are limited. This information provides the basis to understand the impact of different PI on the molecular mechanisms of platelet function. Moreover, these data may serve as basis for future developments of PI technologies for PCs. Further studies should address the impact of both the PI and the storage duration on platelets in PCs because PI may enable the extension of the shelf life of PCs by reducing the bacterial contamination risk. Copyright © 2014 Elsevier Inc. All rights reserved.

Statistical characterization of multiple-reaction monitoring mass spectrometry (MRM-MS) assays for quantitative proteomics

PubMed Central

2012-01-01

Multiple reaction monitoring mass spectrometry (MRM-MS) with stable isotope dilution (SID) is increasingly becoming a widely accepted assay for the quantification of proteins and peptides. These assays have shown great promise in relatively high throughput verification of candidate biomarkers. While the use of MRM-MS assays is well established in the small molecule realm, their introduction and use in proteomics is relatively recent. As such, statistical and computational methods for the analysis of MRM-MS data from proteins and peptides are still being developed. Based on our extensive experience with analyzing a wide range of SID-MRM-MS data, we set forth a methodology for analysis that encompasses significant aspects ranging from data quality assessment, assay characterization including calibration curves, limits of detection (LOD) and quantification (LOQ), and measurement of intra- and interlaboratory precision. We draw upon publicly available seminal datasets to illustrate our methods and algorithms. PMID:23176545

Statistical characterization of multiple-reaction monitoring mass spectrometry (MRM-MS) assays for quantitative proteomics.

PubMed

Mani, D R; Abbatiello, Susan E; Carr, Steven A

2012-01-01

Multiple reaction monitoring mass spectrometry (MRM-MS) with stable isotope dilution (SID) is increasingly becoming a widely accepted assay for the quantification of proteins and peptides. These assays have shown great promise in relatively high throughput verification of candidate biomarkers. While the use of MRM-MS assays is well established in the small molecule realm, their introduction and use in proteomics is relatively recent. As such, statistical and computational methods for the analysis of MRM-MS data from proteins and peptides are still being developed. Based on our extensive experience with analyzing a wide range of SID-MRM-MS data, we set forth a methodology for analysis that encompasses significant aspects ranging from data quality assessment, assay characterization including calibration curves, limits of detection (LOD) and quantification (LOQ), and measurement of intra- and interlaboratory precision. We draw upon publicly available seminal datasets to illustrate our methods and algorithms.

75 FR 77869 - Endocrine Disruptor Screening Program; Second List of Chemicals for Tier 1 Screening; Extension...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-12-14

... ENVIRONMENTAL PROTECTION AGENCY [EPA-HQ-OPPT-2009-0477; FRL-8856-5] Endocrine Disruptor Screening... Federal Register issue of November 17, 2010, concerning the Endocrine Disruptor Screening Program's (EDSP... CONTACT. List of Subjects Environmental protection, Chemicals, Drinking water, Endocrine disruptors...

Pooled-matrix protein interaction screens using Barcode Fusion Genetics.

PubMed

Yachie, Nozomu; Petsalaki, Evangelia; Mellor, Joseph C; Weile, Jochen; Jacob, Yves; Verby, Marta; Ozturk, Sedide B; Li, Siyang; Cote, Atina G; Mosca, Roberto; Knapp, Jennifer J; Ko, Minjeong; Yu, Analyn; Gebbia, Marinella; Sahni, Nidhi; Yi, Song; Tyagi, Tanya; Sheykhkarimli, Dayag; Roth, Jonathan F; Wong, Cassandra; Musa, Louai; Snider, Jamie; Liu, Yi-Chun; Yu, Haiyuan; Braun, Pascal; Stagljar, Igor; Hao, Tong; Calderwood, Michael A; Pelletier, Laurence; Aloy, Patrick; Hill, David E; Vidal, Marc; Roth, Frederick P

2016-04-22

High-throughput binary protein interaction mapping is continuing to extend our understanding of cellular function and disease mechanisms. However, we remain one or two orders of magnitude away from a complete interaction map for humans and other major model organisms. Completion will require screening at substantially larger scales with many complementary assays, requiring further efficiency gains in proteome-scale interaction mapping. Here, we report Barcode Fusion Genetics-Yeast Two-Hybrid (BFG-Y2H), by which a full matrix of protein pairs can be screened in a single multiplexed strain pool. BFG-Y2H uses Cre recombination to fuse DNA barcodes from distinct plasmids, generating chimeric protein-pair barcodes that can be quantified via next-generation sequencing. We applied BFG-Y2H to four different matrices ranging in scale from ~25 K to 2.5 M protein pairs. The results show that BFG-Y2H increases the efficiency of protein matrix screening, with quality that is on par with state-of-the-art Y2H methods. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

Extended Follow-Up Frequently Asked Questions | Division of Cancer Prevention

Cancer.gov

1. What is the PLCO Central Data Coordinating Center?The PLCO Central Data Coordinating Center (CDCC) is an extension of the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial. The extension will be coordinated by one site under the direction of the National Cancer Institute (NCI) rather than ten PLCO Screening Centers to reduce costs. Westat which has served

Early Detection Of Breast Cancer using Post-Translationally Modified Biomarkers

DTIC Science & Technology

2012-03-01

methods are widely used to screen many potential diseases based on changes in blood proteins. Changes in proteins identified by proteomic studies are...suggest that circulating PTM levels can be used as a biomarker for endothelial cell dysfunction, which is of concern in several human diseases . We have...plasmid expressing the wild type PTENP1 3’UTR (pGLU/ψ3’UTR) or the 3’UTR in which the seed matches of the 5 PTEN-targeting microRNAs have been

Identification of cancer protein biomarkers using proteomic techniques

DOEpatents

Mor, Gil G.; Ward, David C.; Bray-Ward, Patricia

2016-10-18

The claimed invention describes methods to diagnose or aid in the diagnosis of cancer. The claimed methods are based on the identification of biomarkers which are particularly well suited to discriminate between cancer subjects and healthy subjects. These biomarkers were identified using a unique and novel screening method described herein. The biomarkers identified herein can also be used in the prognosis and monitoring of cancer. The invention comprises the use of leptin, prolactin, OPN and IGF-II for diagnosing, prognosis and monitoring of ovarian cancer.

Identification of cancer protein biomarkers using proteomic techniques

DOEpatents

Mor, Gil G; Ward, David C; Bray-Ward, Patricia

2015-03-10

The claimed invention describes methods to diagnose or aid in the diagnosis of cancer. The claimed methods are based on the identification of biomarkers which are particularly well suited to discriminate between cancer subjects and healthy subjects. These biomarkers were identified using a unique and novel screening method described herein. The biomarkers identified herein can also be used in the prognosis and monitoring of cancer. The invention comprises the use of leptin, prolactin, OPN and IGF-II for diagnosing, prognosis and monitoring of ovarian cancer.

Identification of cancer protein biomarkers using proteomic techniques

DOEpatents

Mor, Gil G.; Ward, David C.; Bray-Ward, Patricia

2010-02-23

The claimed invention describes methods to diagnose or aid in the diagnosis of cancer. The claimed methods are based on the identification of biomarkers which are particularly well suited to discriminate between cancer subjects and healthy subjects. These biomarkers were identified using a unique and novel screening method described herein. The biomarkers identified herein can also be used in the prognosis and monitoring of cancer. The invention comprises the use of leptin, prolactin, OPN and IGF-II for diagnosing, prognosis and monitoring of ovarian cancer.

Vibrational imaging of newly synthesized proteins in live cells by stimulated Raman scattering microscopy

PubMed Central

Wei, Lu; Yu, Yong; Shen, Yihui; Wang, Meng C.; Min, Wei

2013-01-01

Synthesis of new proteins, a key step in the central dogma of molecular biology, has been a major biological process by which cells respond rapidly to environmental cues in both physiological and pathological conditions. However, the selective visualization of a newly synthesized proteome in living systems with subcellular resolution has proven to be rather challenging, despite the extensive efforts along the lines of fluorescence staining, autoradiography, and mass spectrometry. Herein, we report an imaging technique to visualize nascent proteins by harnessing the emerging stimulated Raman scattering (SRS) microscopy coupled with metabolic incorporation of deuterium-labeled amino acids. As a first demonstration, we imaged newly synthesized proteins in live mammalian cells with high spatial–temporal resolution without fixation or staining. Subcellular compartments with fast protein turnover in HeLa and HEK293T cells, and newly grown neurites in differentiating neuron-like N2A cells, are clearly identified via this imaging technique. Technically, incorporation of deuterium-labeled amino acids is minimally perturbative to live cells, whereas SRS imaging of exogenous carbon–deuterium bonds (C–D) in the cell-silent Raman region is highly sensitive, specific, and compatible with living systems. Moreover, coupled with label-free SRS imaging of the total proteome, our method can readily generate spatial maps of the quantitative ratio between new and total proteomes. Thus, this technique of nonlinear vibrational imaging of stable isotope incorporation will be a valuable tool to advance our understanding of the complex spatial and temporal dynamics of newly synthesized proteome in vivo. PMID:23798434

Organellar proteomics reveals hundreds of novel nuclear proteins in the malaria parasite Plasmodium falciparum

PubMed Central

2012-01-01

Background The post-genomic era of malaria research provided unprecedented insights into the biology of Plasmodium parasites. Due to the large evolutionary distance to model eukaryotes, however, we lack a profound understanding of many processes in Plasmodium biology. One example is the cell nucleus, which controls the parasite genome in a development- and cell cycle-specific manner through mostly unknown mechanisms. To study this important organelle in detail, we conducted an integrative analysis of the P. falciparum nuclear proteome. Results We combined high accuracy mass spectrometry and bioinformatic approaches to present for the first time an experimentally determined core nuclear proteome for P. falciparum. Besides a large number of factors implicated in known nuclear processes, one-third of all detected proteins carry no functional annotation, including many phylum- or genus-specific factors. Importantly, extensive experimental validation using 30 transgenic cell lines confirmed the high specificity of this inventory, and revealed distinct nuclear localization patterns of hitherto uncharacterized proteins. Further, our detailed analysis identified novel protein domains potentially implicated in gene transcription pathways, and sheds important new light on nuclear compartments and processes including regulatory complexes, the nucleolus, nuclear pores, and nuclear import pathways. Conclusion Our study provides comprehensive new insight into the biology of the Plasmodium nucleus and will serve as an important platform for dissecting general and parasite-specific nuclear processes in malaria parasites. Moreover, as the first nuclear proteome characterized in any protist organism, it will provide an important resource for studying evolutionary aspects of nuclear biology. PMID:23181666

Proteomic and phosphoproteomic analysis of polyethylene glycol-induced osmotic stress in root tips of common bean (Phaseolus vulgaris L.)

PubMed Central

Horst, Walter Johannes

2013-01-01

Previous studies have shown that polyethylene glycol (PEG)-induced osmotic stress (OS) reduces cell-wall (CW) porosity and limits aluminium (Al) uptake by root tips of common bean (Phaseolus vulgaris L.). A subsequent transcriptomic study suggested that genes related to CW processes are involved in adjustment to OS. In this study, a proteomic and phosphoproteomic approach was applied to identify OS-induced protein regulation to further improve our understanding of how OS affects Al accumulation. Analysis of total soluble proteins in root tips indicated that, in total, 22 proteins were differentially regulated by OS; these proteins were functionally categorized. Seventy-seven per- cent of the total expressed proteins were involved in metabolic pathways, particularly of carbohydrate and amino acid metabolism. An analysis of the apoplastic proteome revealed that OS reduced the level of five proteins and increased that of seven proteins. Investigation of the total soluble phosphoproteome suggested that dehydrin responded to OS with an enhanced phosphorylation state without a change in abundance. A cellular immunolocalization analysis indicated that dehydrin was localized mainly in the CW. This suggests that dehydrin may play a major protective role in the OS-induced physical breakdown of the CW structure and thus maintenance of the reversibility of CW extensibility during recovery from OS. The proteomic and phosphoproteomic analyses provided novel insights into the complex mechanisms of OS-induced reduction of Al accumulation in the root tips of common bean and highlight a key role for modification of CW structure. PMID:24123251

Symbiosis induces widespread changes in the proteome of the model cnidarian Aiptasia.

PubMed

Oakley, Clinton A; Ameismeier, Michael F; Peng, Lifeng; Weis, Virginia M; Grossman, Arthur R; Davy, Simon K

2016-07-01

Coral reef ecosystems are metabolically founded on the mutualism between corals and photosynthetic dinoflagellates of the genus Symbiodinium. The glass anemone Aiptasia sp. has become a tractable model for this symbiosis, and recent advances in genetic information have enabled the use of mass spectrometry-based proteomics in this model. We utilized label-free liquid chromatography electrospray-ionization tandem mass spectrometry to analyze the effects of symbiosis on the proteomes of symbiotic and aposymbiotic Aiptasia. We identified and obtained relative quantification of more than 3,300 proteins in 1,578 protein clusters, with 81 protein clusters showing significantly different expression between symbiotic states. Symbiotic anemones showed significantly higher expression of proteins involved in lipid storage and transport, nitrogen transport and cycling, intracellular trafficking, endocytosis and inorganic carbon transport. These changes reflect shifts in host metabolism and nutrient reserves due to increased nutritional exchange with the symbionts, as well as mechanisms for supplying inorganic nutrients to the algae. Aposymbiotic anemones exhibited increased expression of multiple systems responsible for mediating reactive oxygen stress, suggesting that the host derives direct or indirect protection from oxidative stress while in symbiosis. Aposymbiotic anemones also increased their expression of an array of proteases and chitinases, indicating a metabolic shift from autotrophy to heterotrophy. These results provide a comprehensive Aiptasia proteome with more direct relative quantification of protein abundance than transcriptomic methods. The extension of "omics" techniques to this model system will allow more powerful studies of coral physiology, ecosystem function, and the effects of biotic and abiotic stress on the coral-dinoflagellate mutualism. © 2015 John Wiley & Sons Ltd.

Comparative Activity-Based Flavin-Dependent Oxidase Profiling.

PubMed

Krysiak, Joanna; Breinbauer, Rolf

2017-01-01

Activity-based protein profiling (ABPP) has become a powerful chemoproteomic technology allowing for the dissection of complex ligand-protein interactions in their native cellular environment. One of the biggest challenges for ABPP is the extension of the proteome coverage. In this chapter a new ABPP strategy dedicated to monoamine oxidases (MAO) is presented. These enzymes are representative examples of flavin-dependent oxidases, playing a crucial role in the regulation of nervous system signaling.

Comparative proteomic profiling of the choline transporter-like1 (CHER1) mutant provides insights into plasmodesmata composition of fully developed Arabidopsis thaliana leaves.

PubMed

Kraner, Max E; Müller, Carmen; Sonnewald, Uwe

2017-11-01

In plants, intercellular communication and exchange are highly dependent on cell wall bridging structures between adhering cells, so-called plasmodesmata (PD). In our previous genetic screen for PD-deficient Arabidopsis mutants, we described choline transporter-like 1 (CHER1) being important for PD genesis and maturation. Leaves of cher1 mutant plants have up to 10 times less PD, which do not develop to complex structures. Here we utilize the T-DNA insertion mutant cher1-4 and report a deep comparative proteomic workflow for the identification of cell-wall-embedded PD-associated proteins. Analyzing triplicates of cell-wall-enriched fractions in depth by fractionation and quantitative high-resolution mass spectrometry, we compared > 5000 proteins obtained from fully developed leaves. Comparative data analysis and subsequent filtering generated a list of 61 proteins being significantly more abundant in Col-0. This list was enriched for previously described PD-associated proteins. To validate PD association of so far uncharacterized proteins, subcellular localization analyses were carried out by confocal laser-scanning microscopy. This study confirmed the association of PD for three out of four selected candidates, indicating that the comparative approach indeed allowed identification of so far undescribed PD-associated proteins. Performing comparative cell wall proteomics of Nicotiana benthamiana tissue, we observed an increase in abundance of these three selected candidates during sink to source transition. Taken together, our comparative proteomic approach revealed a valuable data set of potential PD-associated proteins, which can be used as a resource to unravel the molecular composition of complex PD and to investigate their function in cell-to-cell communication. © 2017 The Authors. The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

Identification of proteins capable of metal reduction from the proteome of the Gram-positive bacterium Desulfotomaculum reducens MI-1 using an NADH-based activity assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Otwell, Annie E.; Sherwood, Roberts; Zhang, Sheng

Metal reduction capability has been found in numerous species of environmentally abundant Gram-positive bacteria. However, understanding of microbial metal reduction is based almost solely on studies of Gram-negative organisms. In this study, we focus on Desulfotomaculum reducens MI-1, a Gram-positive metal reducer whose genome lacks genes with similarity to any characterized metal reductase. D. reducens has been shown to reduce not only Fe(III), but also the environmentally important contaminants U(VI) and Cr(VI). By extracting, separating, and analyzing the functional proteome of D. reducens, using a ferrozine-based assay in order to screen for chelated Fe(III)-NTA reduction with NADH as electron donor,more » we have identified proteins not previously characterized as iron reductases. Their function was confirmed by heterologous expression in E. coli. These are the protein NADH:flavin oxidoreductase (Dred_2421) and a protein complex composed of oxidoreductase FAD/NAD(P)-binding subunit (Dred_1685) and dihydroorotate dehydrogenase 1B (Dred_1686). Dred_2421 was identified in the soluble proteome and is predicted to be a cytoplasmic protein. Dred_1685 and Dred_1686 were identified in both the soluble as well as the insoluble (presumably membrane) protein fraction, suggesting a type of membrane-association, although PSORTb predicts both proteins are cytoplasmic. Furthermore, we show that these proteins have the capability to reduce soluble Cr(VI) and U(VI) with NADH as electron donor. This study is the first functional proteomic analysis of D. reducens, and one of the first analyses of metal and radionuclide reduction in an environmentally relevant Gram-positive bacterium.« less

Protein differences between human trapezius and vastus lateralis muscles determined with a proteomic approach.

PubMed

Hadrévi, Jenny; Hellström, Fredrik; Kieselbach, Thomas; Malm, Christer; Pedrosa-Domellöf, Fatima

2011-08-10

The trapezius muscle is a neck muscle that is susceptible to chronic pain conditions associated with repetitive tasks, commonly referred to as chronic work-related myalgia, hence making the trapezius a muscle of clinical interest. To provide a basis for further investigations of the proteomic traits of the trapezius muscle in disease, two-dimensional difference gel electrophoresis (2D-DIGE) was performed on the healthy trapezius using vastus lateralis as a reference. To obtain as much information as possible from the vast proteomic data set, both one-way ANOVA, with and without false discovery rate (FDR) correlation, and partial least square projection to latent structures with discriminant analysis (PLS-DA) were combined to compare the outcome of the analysis. The trapezius and vastus lateralis showed significant differences in metabolic, contractile and regulatory proteins, with different results depending on choice of statistical approach and pre-processing technique. Using the standard method, FDR correlated one-way ANOVA, 42 protein spots differed significantly in abundance between the two muscles. Complementary analysis using immunohistochemistry and western blot confirmed the results from the 2D-DIGE analysis. The proteomic approach used in the present study combining 2D-DIGE and multivariate modelling provided a more comprehensive comparison of the protein profiles of the human trapezius and vastus lateralis muscle, than previously possible to obtain with immunohistochemistry or SDS-PAGE alone. Although 2D-DIGE has inherent limitations it is particularly useful to comprehensively screen for important structural and metabolic proteins, and appears to be a promising tool for future studies of patients suffering from chronic work related myalgia or other muscle diseases.

SWATH™- and iTRAQ-based quantitative proteomic analyses reveal an overexpression and biological relevance of CD109 in advanced NSCLC.

PubMed

Zhang, Fanglin; Lin, Hechun; Gu, Aiqin; Li, Jing; Liu, Lei; Yu, Tao; Cui, Yongqi; Deng, Wei; Yan, Mingxia; Li, Jinjun; Yao, Ming

2014-05-06

To identify cancer-related proteins, we used isobaric tags in a relative and absolute quantitation (iTRAQ) proteomic approach and SWATH™ quantification approach to analyze the secretome of an isogenic pair of highly metastatic and low metastatic non-small-cell lung cancer (NSCLC) cell lines. In addition, we compared two groups of pooled serum samples (12 early-stage and 12 late-stage patients) to mine data for candidates screened by iTRAQ-labeled proteomic analysis. A total of 110 proteins and 71 proteins were observed to be significantly differentially expressed in the cell line secretome and NSCLC sera, respectively. Among these proteins, CD109 was found to be highly expressed in both the highly metastatic cell line secretome and the group of late-stage patients. A sandwich ELISA assay also demonstrated an elevation of serum CD109 levels in individual NSCLC patients (n=30) compared with healthy subjects (n=19). Furthermore, CD109 displayed higher expression in lung cancer tissues compared with their matched noncancerous lung tissues (n=72). In addition, the knockdown of CD109 influenced several NSCLC cell bio-functions, for instance, depressing cell growth, affecting cell cycle phases. These phenomena suggest that CD109 plays a critical role in NSCLC progression. We simultaneously applied two quantitative proteomic approaches-iTRAQ-labeling and SWATH™-to analyze the secretome of metastatic cell lines, in order to explore the cancer-associated proteins in conditioned media. In this study, our results indicate that CD109 plays a critical role in non-small-cell lung cancer (NSCLC) progression, and is overexpressed in advanced NSCLC. Copyright © 2014 Elsevier B.V. All rights reserved.

Early Targets of miR-34a in Neuroblastoma*

PubMed Central

De Antonellis, Pasqualino; Carotenuto, Marianeve; Vandenbussche, Jonathan; De Vita, Gennaro; Ferrucci, Veronica; Medaglia, Chiara; Boffa, Iolanda; Galiero, Alessandra; Di Somma, Sarah; Magliulo, Daniela; Aiese, Nadia; Alonzi, Alessandro; Spano, Daniela; Liguori, Lucia; Chiarolla, Cristina; Verrico, Antonio; Schulte, Johannes H.; Mestdagh, Pieter; Vandesompele, Jo; Gevaert, Kris; Zollo, Massimo

2014-01-01

Several genes encoding for proteins involved in proliferation, invasion, and apoptosis are known to be direct miR-34a targets. Here, we used proteomics to screen for targets of miR-34a in neuroblastoma (NBL), a childhood cancer that originates from precursor cells of the sympathetic nervous system. We examined the effect of miR-34a overexpression using a tetracycline inducible system in two NBL cell lines (SHEP and SH-SY5Y) at early time points of expression (6, 12, and 24 h). Proteome analysis using post-metabolic labeling led to the identification of 2,082 proteins, and among these 186 were regulated (112 proteins down-regulated and 74 up-regulated). Prediction of miR-34a targets via bioinformatics showed that 32 transcripts held miR-34a seed sequences in their 3′-UTR. By combining the proteomics data with Kaplan Meier gene-expression studies, we identified seven new gene products (ALG13, TIMM13, TGM2, ABCF2, CTCF, Ki67, and LYAR) that were correlated with worse clinical outcomes. These were further validated in vitro by 3′-UTR seed sequence regulation. In addition, Michigan Molecular Interactions searches indicated that together these proteins affect signaling pathways that regulate cell cycle and proliferation, focal adhesions, and other cellular properties that overall enhance tumor progression (including signaling pathways such as TGF-β, WNT, MAPK, and FAK). In conclusion, proteome analysis has here identified early targets of miR-34a with relevance to NBL tumorigenesis. Along with the results of previous studies, our data strongly suggest miR-34a as a useful tool for improving the chance of therapeutic success with NBL. PMID:24912852

Identification of p90 Ribosomal S6 Kinase 2 as a Novel Host Protein in HBx Augmenting HBV Replication by iTRAQ-Based Quantitative Comparative Proteomics.

PubMed

Yan, Li-Bo; Yu, You-Jia; Zhang, Qing-Bo; Tang, Xiao-Qiong; Bai, Lang; Huang, FeiJun; Tang, Hong

2018-05-01

The aim of this study was to screen for novel host proteins that play a role in HBx augmenting Hepatitis B virus (HBV) replication. Three HepG2 cell lines stably harboring different functional domains of HBx (HBx, HBx-Cm6, and HBx-Cm16) were cultured. ITRAQ technology integrated with LC-MS/MS analysis was applied to identify the proteome differences among these three cell lines. In brief, a total of 70 different proteins were identified among HepG2-HBx, HepG2-HBx-Cm6, and HepG2-HBx-Cm16 by double repetition. Several differentially expressed proteins, including p90 ribosomal S6 kinase 2 (RSK2), were further validated. RSK2 was expressed at higher levels in HepG2-HBx and HepG2-HBx-Cm6 compared with HepG2-HBx-Cm16. Furthermore, levels of HBV replication intermediates were decreased after silencing RSK2 in HepG2.2.15. An HBx-minus HBV mutant genome led to decreased levels of HBV replication intermediates and these decreases were restored to levels similar to wild-type HBV by transient ectopic expression of HBx. After silencing RSK2 expression, the levels of HBV replication intermediates synthesized from the HBx-minus HBV mutant genome were not restored to levels that were observed with wild-type HBV by transient HBx expression. Based on iTRAQ quantitative comparative proteomics, RSK2 was identified as a novel host protein that plays a role in HBx augmenting HBV replication. © 2018 The Authors. Proteomics - Clinical Application Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

18O-labeled proteome reference as global internal standards for targeted quantification by selected reaction monitoring-mass spectrometry.

PubMed

Kim, Jong-Seo; Fillmore, Thomas L; Liu, Tao; Robinson, Errol; Hossain, Mahmud; Champion, Boyd L; Moore, Ronald J; Camp, David G; Smith, Richard D; Qian, Wei-Jun

2011-12-01

Selected reaction monitoring (SRM)-MS is an emerging technology for high throughput targeted protein quantification and verification in biomarker discovery studies; however, the cost associated with the application of stable isotope-labeled synthetic peptides as internal standards can be prohibitive for screening a large number of candidate proteins as often required in the preverification phase of discovery studies. Herein we present a proof of concept study using an (18)O-labeled proteome reference as global internal standards (GIS) for SRM-based relative quantification. The (18)O-labeled proteome reference (or GIS) can be readily prepared and contains a heavy isotope ((18)O)-labeled internal standard for every possible tryptic peptide. Our results showed that the percentage of heavy isotope ((18)O) incorporation applying an improved protocol was >99.5% for most peptides investigated. The accuracy, reproducibility, and linear dynamic range of quantification were further assessed based on known ratios of standard proteins spiked into the labeled mouse plasma reference. Reliable quantification was observed with high reproducibility (i.e. coefficient of variance <10%) for analyte concentrations that were set at 100-fold higher or lower than those of the GIS based on the light ((16)O)/heavy ((18)O) peak area ratios. The utility of (18)O-labeled GIS was further illustrated by accurate relative quantification of 45 major human plasma proteins. Moreover, quantification of the concentrations of C-reactive protein and prostate-specific antigen was illustrated by coupling the GIS with standard additions of purified protein standards. Collectively, our results demonstrated that the use of (18)O-labeled proteome reference as GIS provides a convenient, low cost, and effective strategy for relative quantification of a large number of candidate proteins in biological or clinical samples using SRM.

PNAC: a protein nucleolar association classifier

PubMed Central

2011-01-01

Background Although primarily known as the site of ribosome subunit production, the nucleolus is involved in numerous and diverse cellular processes. Recent large-scale proteomics projects have identified thousands of human proteins that associate with the nucleolus. However, in most cases, we know neither the fraction of each protein pool that is nucleolus-associated nor whether their association is permanent or conditional. Results To describe the dynamic localisation of proteins in the nucleolus, we investigated the extent of nucleolar association of proteins by first collating an extensively curated literature-derived dataset. This dataset then served to train a probabilistic predictor which integrates gene and protein characteristics. Unlike most previous experimental and computational studies of the nucleolar proteome that produce large static lists of nucleolar proteins regardless of their extent of nucleolar association, our predictor models the fluidity of the nucleolus by considering different classes of nucleolar-associated proteins. The new method predicts all human proteins as either nucleolar-enriched, nucleolar-nucleoplasmic, nucleolar-cytoplasmic or non-nucleolar. Leave-one-out cross validation tests reveal sensitivity values for these four classes ranging from 0.72 to 0.90 and positive predictive values ranging from 0.63 to 0.94. The overall accuracy of the classifier was measured to be 0.85 on an independent literature-based test set and 0.74 using a large independent quantitative proteomics dataset. While the three nucleolar-association groups display vastly different Gene Ontology biological process signatures and evolutionary characteristics, they collectively represent the most well characterised nucleolar functions. Conclusions Our proteome-wide classification of nucleolar association provides a novel representation of the dynamic content of the nucleolus. This model of nucleolar localisation thus increases the coverage while providing accurate and specific annotations of the nucleolar proteome. It will be instrumental in better understanding the central role of the nucleolus in the cell and its interaction with other subcellular compartments. PMID:21272300

PeptidePicker: a scientific workflow with web interface for selecting appropriate peptides for targeted proteomics experiments.

PubMed

Mohammed, Yassene; Domański, Dominik; Jackson, Angela M; Smith, Derek S; Deelder, André M; Palmblad, Magnus; Borchers, Christoph H

2014-06-25

One challenge in Multiple Reaction Monitoring (MRM)-based proteomics is to select the most appropriate surrogate peptides to represent a target protein. We present here a software package to automatically generate these most appropriate surrogate peptides for an LC/MRM-MS analysis. Our method integrates information about the proteins, their tryptic peptides, and the suitability of these peptides for MRM which is available online in UniProtKB, NCBI's dbSNP, ExPASy, PeptideAtlas, PRIDE, and GPMDB. The scoring algorithm reflects our knowledge in choosing the best candidate peptides for MRM, based on the uniqueness of the peptide in the targeted proteome, its physiochemical properties, and whether it previously has been observed. The modularity of the workflow allows further extension and additional selection criteria to be incorporated. We have developed a simple Web interface where the researcher provides the protein accession number, the subject organism, and peptide-specific options. Currently, the software is designed for human and mouse proteomes, but additional species can be easily be added. Our software improved the peptide selection by eliminating human error, considering multiple data sources and all of the isoforms of the protein, and resulted in faster peptide selection - approximately 50 proteins per hour compared to 8 per day. Compiling a list of optimal surrogate peptides for target proteins to be analyzed by LC/MRM-MS has been a cumbersome process, in which expert researchers retrieved information from different online repositories and used their own reasoning to find the most appropriate peptides. Our scientific workflow automates this process by integrating information from different data sources including UniProt, Global Proteome Machine, NCBI's dbSNP, and PeptideAtlas, simulating the researchers' reasoning, and incorporating their knowledge of how to select the best proteotypic peptides for an MRM analysis. The developed software can help to standardize the selection of peptides, eliminate human error, and increase productivity. Copyright © 2014 Elsevier B.V. All rights reserved.

Assessment and improvement of statistical tools for comparative proteomics analysis of sparse data sets with few experimental replicates.

PubMed

Schwämmle, Veit; León, Ileana Rodríguez; Jensen, Ole Nørregaard

2013-09-06

Large-scale quantitative analyses of biological systems are often performed with few replicate experiments, leading to multiple nonidentical data sets due to missing values. For example, mass spectrometry driven proteomics experiments are frequently performed with few biological or technical replicates due to sample-scarcity or due to duty-cycle or sensitivity constraints, or limited capacity of the available instrumentation, leading to incomplete results where detection of significant feature changes becomes a challenge. This problem is further exacerbated for the detection of significant changes on the peptide level, for example, in phospho-proteomics experiments. In order to assess the extent of this problem and the implications for large-scale proteome analysis, we investigated and optimized the performance of three statistical approaches by using simulated and experimental data sets with varying numbers of missing values. We applied three tools, including standard t test, moderated t test, also known as limma, and rank products for the detection of significantly changing features in simulated and experimental proteomics data sets with missing values. The rank product method was improved to work with data sets containing missing values. Extensive analysis of simulated and experimental data sets revealed that the performance of the statistical analysis tools depended on simple properties of the data sets. High-confidence results were obtained by using the limma and rank products methods for analyses of triplicate data sets that exhibited more than 1000 features and more than 50% missing values. The maximum number of differentially represented features was identified by using limma and rank products methods in a complementary manner. We therefore recommend combined usage of these methods as a novel and optimal way to detect significantly changing features in these data sets. This approach is suitable for large quantitative data sets from stable isotope labeling and mass spectrometry experiments and should be applicable to large data sets of any type. An R script that implements the improved rank products algorithm and the combined analysis is available.

Analysis of the pumpkin phloem proteome provides insights into angiosperm sieve tube function.

PubMed

Lin, Ming-Kuem; Lee, Young-Jin; Lough, Tony J; Phinney, Brett S; Lucas, William J

2009-02-01

Increasing evidence suggests that proteins present in the angiosperm sieve tube system play an important role in the long distance signaling system of plants. To identify the nature of these putatively non-cell-autonomous proteins, we adopted a large scale proteomics approach to analyze pumpkin phloem exudates. Phloem proteins were fractionated by fast protein liquid chromatography using both anion and cation exchange columns and then either in-solution or in-gel digested following further separation by SDS-PAGE. A total of 345 LC-MS/MS data sets were analyzed using a combination of Mascot and X!Tandem against the NCBI non-redundant green plant database and an extensive Cucurbit maxima expressed sequence tag database. In this analysis, 1,209 different consensi were obtained of which 1,121 could be annotated from GenBank and BLAST search analyses against three plant species, Arabidopsis thaliana, rice (Oryza sativa), and poplar (Populus trichocarpa). Gene ontology (GO) enrichment analyses identified sets of phloem proteins that function in RNA binding, mRNA translation, ubiquitin-mediated proteolysis, and macromolecular and vesicle trafficking. Our findings indicate that protein synthesis and turnover, processes that were thought to be absent in enucleate sieve elements, likely occur within the angiosperm phloem translocation stream. In addition, our GO analysis identified a set of phloem proteins that are associated with the GO term "embryonic development ending in seed dormancy"; this finding raises the intriguing question as to whether the phloem may exert some level of control over seed development. The universal significance of the phloem proteome was highlighted by conservation of the phloem proteome in species as diverse as monocots (rice), eudicots (Arabidopsis and pumpkin), and trees (poplar). These results are discussed from the perspective of the role played by the phloem proteome as an integral component of the whole plant communication system.

Knowledge-driven lead discovery.

PubMed

Pirard, Bernard

2005-11-01

Virtual screening encompasses several computational approaches which have proven valuable for identifying novel leads. These approaches rely on available information. Herein, we review recent successful applications of virtual screening. The extension of virtual screening methodologies to target families is also briefly discussed.

Proteomic Biomarkers for Acute Interstitial Lung Disease in Gefitinib-Treated Japanese Lung Cancer Patients

PubMed Central

Kawakami, Takao; Nagasaka, Keiko; Takami, Sachiko; Wada, Kazuya; Tu, Hsiao-Kun; Otsuji, Makiko; Kyono, Yutaka; Dobashi, Tae; Komatsu, Yasuhiko; Kihara, Makoto; Akimoto, Shingo; Peers, Ian S.; South, Marie C.; Higenbottam, Tim; Fukuoka, Masahiro; Nakata, Koichiro; Ohe, Yuichiro; Kudoh, Shoji; Clausen, Ib Groth; Nishimura, Toshihide; Marko-Varga, György; Kato, Harubumi

2011-01-01

Interstitial lung disease (ILD) events have been reported in Japanese non-small-cell lung cancer (NSCLC) patients receiving EGFR tyrosine kinase inhibitors. We investigated proteomic biomarkers for mechanistic insights and improved prediction of ILD. Blood plasma was collected from 43 gefitinib-treated NSCLC patients developing acute ILD (confirmed by blinded diagnostic review) and 123 randomly selected controls in a nested case-control study within a pharmacoepidemiological cohort study in Japan. We generated ∼7 million tandem mass spectrometry (MS/MS) measurements with extensive quality control and validation, producing one of the largest proteomic lung cancer datasets to date, incorporating rigorous study design, phenotype definition, and evaluation of sample processing. After alignment, scaling, and measurement batch adjustment, we identified 41 peptide peaks representing 29 proteins best predicting ILD. Multivariate peptide, protein, and pathway modeling achieved ILD prediction comparable to previously identified clinical variables; combining the two provided some improvement. The acute phase response pathway was strongly represented (17 of 29 proteins, p = 1.0×10−25), suggesting a key role with potential utility as a marker for increased risk of acute ILD events. Validation by Western blotting showed correlation for identified proteins, confirming that robust results can be generated from an MS/MS platform implementing strict quality control. PMID:21799770

Comparative physiological and proteomic analyses reveal the actions of melatonin in the reduction of oxidative stress in Bermuda grass (Cynodon dactylon (L). Pers.).

PubMed

Shi, Haitao; Wang, Xin; Tan, Dun-Xian; Reiter, Russel J; Chan, Zhulong

2015-08-01

The fact of melatonin as an important antioxidant in animals led plant researchers to speculate that melatonin also acts in the similar manner in plants. Although melatonin has significant effects on alleviating stress-triggered reactive oxygen species (ROS), the involvement of melatonin in direct oxidative stress and the underlying physiological and molecular mechanisms remain unclear in plants. In this study, we found that exogenous melatonin significantly alleviated hydrogen peroxide (H2O2)-modulated plant growth, cell damage, and ROS accumulation in Bermuda grass. Additionally, 76 proteins significantly influenced by melatonin during mock or H2O2 treatment were identified by gel-free proteomics using iTRAQ (isobaric tags for relative and absolute quantitation). Metabolic pathway analysis showed that several pathways were markedly enhanced by melatonin and H2O2 treatments, including polyamine metabolism, ribosome pathway, major carbohydrate metabolism, photosynthesis, redox, and amino acid metabolism. Taken together, this study provides more comprehensive insights into the physiological and molecular mechanisms of melatonin in Bermuda grass responses to direct oxidative stress. This may relate to the activation of antioxidants, modulation of metabolic pathways, and extensive proteome reprograming. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A new reference implementation of the PSICQUIC web service.

PubMed

del-Toro, Noemi; Dumousseau, Marine; Orchard, Sandra; Jimenez, Rafael C; Galeota, Eugenia; Launay, Guillaume; Goll, Johannes; Breuer, Karin; Ono, Keiichiro; Salwinski, Lukasz; Hermjakob, Henning

2013-07-01

The Proteomics Standard Initiative Common QUery InterfaCe (PSICQUIC) specification was created by the Human Proteome Organization Proteomics Standards Initiative (HUPO-PSI) to enable computational access to molecular-interaction data resources by means of a standard Web Service and query language. Currently providing >150 million binary interaction evidences from 28 servers globally, the PSICQUIC interface allows the concurrent search of multiple molecular-interaction information resources using a single query. Here, we present an extension of the PSICQUIC specification (version 1.3), which has been released to be compliant with the enhanced standards in molecular interactions. The new release also includes a new reference implementation of the PSICQUIC server available to the data providers. It offers augmented web service capabilities and improves the user experience. PSICQUIC has been running for almost 5 years, with a user base growing from only 4 data providers to 28 (April 2013) allowing access to 151 310 109 binary interactions. The power of this web service is shown in PSICQUIC View web application, an example of how to simultaneously query, browse and download results from the different PSICQUIC servers. This application is free and open to all users with no login requirement (http://www.ebi.ac.uk/Tools/webservices/psicquic/view/main.xhtml).

Ursgal, Universal Python Module Combining Common Bottom-Up Proteomics Tools for Large-Scale Analysis.

PubMed

Kremer, Lukas P M; Leufken, Johannes; Oyunchimeg, Purevdulam; Schulze, Stefan; Fufezan, Christian

2016-03-04

Proteomics data integration has become a broad field with a variety of programs offering innovative algorithms to analyze increasing amounts of data. Unfortunately, this software diversity leads to many problems as soon as the data is analyzed using more than one algorithm for the same task. Although it was shown that the combination of multiple peptide identification algorithms yields more robust results, it is only recently that unified approaches are emerging; however, workflows that, for example, aim to optimize search parameters or that employ cascaded style searches can only be made accessible if data analysis becomes not only unified but also and most importantly scriptable. Here we introduce Ursgal, a Python interface to many commonly used bottom-up proteomics tools and to additional auxiliary programs. Complex workflows can thus be composed using the Python scripting language using a few lines of code. Ursgal is easily extensible, and we have made several database search engines (X!Tandem, OMSSA, MS-GF+, Myrimatch, MS Amanda), statistical postprocessing algorithms (qvality, Percolator), and one algorithm that combines statistically postprocessed outputs from multiple search engines ("combined FDR") accessible as an interface in Python. Furthermore, we have implemented a new algorithm ("combined PEP") that combines multiple search engines employing elements of "combined FDR", PeptideShaker, and Bayes' theorem.

Elevated temperature alters proteomic responses of individual organisms within a biofilm community

DOE PAGES

Mosier, Annika C.; Li, Zhou; Thomas, Brian C.; ...

2014-07-22

Microbial communities that underpin global biogeochemical cycles will likely be influenced by elevated temperature associated with environmental change. In this paper, we test an approach to measure how elevated temperature impacts the physiology of individual microbial groups in a community context, using a model microbial-based ecosystem. The study is the first application of tandem mass tag (TMT)-based proteomics to a microbial community. We accurately, precisely and reproducibly quantified thousands of proteins in biofilms growing at 40, 43 and 46 °C. Elevated temperature led to upregulation of proteins involved in amino-acid metabolism at the level of individual organisms and the entiremore » community. Proteins from related organisms differed in their relative abundance and functional responses to temperature. Elevated temperature repressed carbon fixation proteins from two Leptospirillum genotypes, whereas carbon fixation proteins were significantly upregulated at higher temperature by a third member of this genus. Leptospirillum group III bacteria may have been subject to viral stress at elevated temperature, which could lead to greater carbon turnover in the microbial food web through the release of viral lysate. Finally, overall, these findings highlight the utility of proteomics-enabled community-based physiology studies, and provide a methodological framework for possible extension to additional mixed culture and environmental sample analyses.« less

A Catalog of Proteins Expressed in the AG Secreted Fluid during the Mature Phase of the Chinese Mitten Crabs (Eriocheir sinensis)

PubMed Central

He, Lin; Li, Qing; Liu, Lihua; Wang, Yuanli; Xie, Jing; Yang, Hongdan; Wang, Qun

2015-01-01

The accessory gland (AG) is an important component of the male reproductive system of arthropods, its secretions enhance fertility, some AG proteins bind to the spermatozoa and affect its function and properties. Here we report the first comprehensive catalog of the AG secreted fluid during the mature phase of the Chinese mitten crab (Eriocheir sinensis). AG proteins were separated by one-dimensional gel electrophoresis and analyzed by reverse phase high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). Altogether, the mass spectra of 1173 peptides were detected (1067 without decoy and contaminants) which allowed for the identification of 486 different proteins annotated upon the NCBI database (http://www.ncbi.nlm.nih.gov/) and our transcritptome dataset. The mass spectrometry proteomics data have been deposited at the ProteomeXchange with identifier PXD000700. An extensive description of the AG proteome will help provide the basis for a better understanding of a number of reproductive mechanisms, including potentially spermatophore breakdown, dynamic functional and morphological changes in sperm cells and sperm acrosin enzyme vitality. Thus, the comprehensive catalog of proteins presented here can serve as a valuable reference for future studies of sperm maturation and regulatory mechanisms involved in crustacean reproduction. PMID:26305468

Quantitative proteomics of the human skin secretome reveal a reduction in immune defense mediators in ectodermal dysplasia patients.

PubMed

Burian, Marc; Velic, Ana; Matic, Katarina; Günther, Stephanie; Kraft, Beatrice; Gonser, Lena; Forchhammer, Stephan; Tiffert, Yvonne; Naumer, Christian; Krohn, Michael; Berneburg, Mark; Yazdi, Amir S; Maček, Boris; Schittek, Birgit

2015-03-01

In healthy human skin host defense molecules such as antimicrobial peptides (AMPs) contribute to skin immune homeostasis. In patients with the congenital disease ectodermal dysplasia (ED) skin integrity is disturbed and as a result patients have recurrent skin infections. The disease is characterized by developmental abnormalities of ectodermal derivatives and absent or reduced sweating. We hypothesized that ED patients have a reduced skin immune defense because of the reduced ability to sweat. Therefore, we performed a label-free quantitative proteome analysis of wash solution of human skin from ED patients or healthy individuals. A clear-cut difference between both cohorts could be observed in cellular processes related to immunity and host defense. In line with the extensive underrepresentation of proteins of the immune system, dermcidin, a sweat-derived AMP, was reduced in its abundance in the skin secretome of ED patients. In contrast, proteins involved in metabolic/catabolic and biosynthetic processes were enriched in the skin secretome of ED patients. In summary, our proteome profiling provides insights into the actual situation of healthy versus diseased skin. The systematic reduction in immune system and defense-related proteins may contribute to the high susceptibility of ED patients to skin infections and altered skin colonization.

Proteogenomics connects somatic mutations to signalling in breast cancer.

PubMed

Mertins, Philipp; Mani, D R; Ruggles, Kelly V; Gillette, Michael A; Clauser, Karl R; Wang, Pei; Wang, Xianlong; Qiao, Jana W; Cao, Song; Petralia, Francesca; Kawaler, Emily; Mundt, Filip; Krug, Karsten; Tu, Zhidong; Lei, Jonathan T; Gatza, Michael L; Wilkerson, Matthew; Perou, Charles M; Yellapantula, Venkata; Huang, Kuan-lin; Lin, Chenwei; McLellan, Michael D; Yan, Ping; Davies, Sherri R; Townsend, R Reid; Skates, Steven J; Wang, Jing; Zhang, Bing; Kinsinger, Christopher R; Mesri, Mehdi; Rodriguez, Henry; Ding, Li; Paulovich, Amanda G; Fenyö, David; Ellis, Matthew J; Carr, Steven A

2016-06-02

Somatic mutations have been extensively characterized in breast cancer, but the effects of these genetic alterations on the proteomic landscape remain poorly understood. Here we describe quantitative mass-spectrometry-based proteomic and phosphoproteomic analyses of 105 genomically annotated breast cancers, of which 77 provided high-quality data. Integrated analyses provided insights into the somatic cancer genome including the consequences of chromosomal loss, such as the 5q deletion characteristic of basal-like breast cancer. Interrogation of the 5q trans-effects against the Library of Integrated Network-based Cellular Signatures, connected loss of CETN3 and SKP1 to elevated expression of epidermal growth factor receptor (EGFR), and SKP1 loss also to increased SRC tyrosine kinase. Global proteomic data confirmed a stromal-enriched group of proteins in addition to basal and luminal clusters, and pathway analysis of the phosphoproteome identified a G-protein-coupled receptor cluster that was not readily identified at the mRNA level. In addition to ERBB2, other amplicon-associated highly phosphorylated kinases were identified, including CDK12, PAK1, PTK2, RIPK2 and TLK2. We demonstrate that proteogenomic analysis of breast cancer elucidates the functional consequences of somatic mutations, narrows candidate nominations for driver genes within large deletions and amplified regions, and identifies therapeutic targets.

Targeted Quantitative Screening of Chromosome 18 Encoded Proteome in Plasma Samples of Astronaut Candidates.

PubMed

Kopylov, Artur T; Ilgisonis, Ekaterina V; Moysa, Alexander A; Tikhonova, Olga V; Zavialova, Maria G; Novikova, Svetlana E; Lisitsa, Andrey V; Ponomarenko, Elena A; Moshkovskii, Sergei A; Markin, Andrey A; Grigoriev, Anatoly I; Zgoda, Victor G; Archakov, Alexander I

2016-11-04

This work was aimed at estimating the concentrations of proteins encoded by human chromosome 18 (Chr 18) in plasma samples of 54 healthy male volunteers (aged 20-47). These young persons have been certified by the medical evaluation board as healthy subjects ready for space flight training. Over 260 stable isotope-labeled peptide standards (SIS) were synthesized to perform the measurements of proteins encoded by Chr 18. Selected reaction monitoring (SRM) with SIS allowed an estimate of the levels of 84 of 276 proteins encoded by Chr 18. These proteins were quantified in whole and depleted plasma samples. Concentration of the proteins detected varied from 10 -6 M (transthyretin, P02766) to 10 -11 M (P4-ATPase, O43861). A minor part of the proteins (mostly representing intracellular proteins) was characterized by extremely high inter individual variations. The results provide a background for studies of a potential biomarker in plasma among proteins encoded by Chr 18. The SRM raw data are available in ProteomeXchange repository (PXD004374).

Comparative analysis of phytochemicals and nutrient availability in two contrasting cultivars of sweet potato (Ipomoea batatas L.).

PubMed

Shekhar, Shubhendu; Mishra, Divya; Buragohain, Alak Kumar; Chakraborty, Subhra; Chakraborty, Niranjan

2015-04-15

Sweet potato ranks as the world's seventh most important food crop, and has major contribution to energy and phytochemical source of nutrition. To unravel the molecular basis for differential nutrient availability, and to exploit the natural genetic variation(s) of sweet potato, a series of physiochemical and proteomics experiment was conducted using two contrasting cultivars, an orange-fleshed sweet potato (OFSP) and a white-fleshed sweet potato (WFSP). Phytochemical screening revealed high percentage of carbohydrate, reducing sugar and phenolics in WFSP, whereas OFSP showed increased levels of total protein, flavonoids, anthocyanins, and carotenoids. The rate of starch and cellulose degradation was found to be less in OFSP during storage, indicating tight regulation of gene(s) responsible for starch-degradation. Comparative proteomics displayed a cultivar-dependent expression of proteins along with evolutionarily conserved proteins. These results suggest that cultivar-specific expression of proteins and/or their interacting partners might play a crucial role for nutrient acquisition in sweet potato. Copyright © 2014 Elsevier Ltd. All rights reserved.

Targeting RAS-driven human cancer cells with antibodies to upregulated and essential cell-surface proteins.

PubMed

Martinko, Alexander J; Truillet, Charles; Julien, Olivier; Diaz, Juan E; Horlbeck, Max A; Whiteley, Gordon; Blonder, Josip; Weissman, Jonathan S; Bandyopadhyay, Sourav; Evans, Michael J; Wells, James A

2018-01-23

While there have been tremendous efforts to target oncogenic RAS signaling from inside the cell, little effort has focused on the cell-surface. Here, we used quantitative surface proteomics to reveal a signature of proteins that are upregulated on cells transformed with KRAS G12V , and driven by MAPK pathway signaling. We next generated a toolkit of recombinant antibodies to seven of these RAS-induced proteins. We found that five of these proteins are broadly distributed on cancer cell lines harboring RAS mutations. In parallel, a cell-surface CRISPRi screen identified integrin and Wnt signaling proteins as critical to RAS-transformed cells. We show that antibodies targeting CDCP1, a protein common to our proteomics and CRISPRi datasets, can be leveraged to deliver cytotoxic and immunotherapeutic payloads to RAS-transformed cancer cells and report for RAS signaling status in vivo. Taken together, this work presents a technological platform for attacking RAS from outside the cell. © 2018, Martinko et al.

A cell death assay for assessing the mitochondrial targeting of proteins.

PubMed

Camara Teixeira, Daniel; Cordonier, Elizabeth L; Wijeratne, Subhashinee S K; Huebbe, Patricia; Jamin, Augusta; Jarecke, Sarah; Wiebe, Matthew; Zempleni, Janos

2018-06-01

The mitochondrial proteome comprises 1000 to 1500 proteins, in addition to proteins for which the mitochondrial localization is uncertain. About 800 diseases have been linked with mutations in mitochondrial proteins. We devised a cell survival assay for assessing the mitochondrial localization in a high-throughput format. This protocol allows us to assess the mitochondrial localization of proteins and their mutants, and to identify drugs and nutrients that modulate the mitochondrial targeting of proteins. The assay works equally well for proteins directed to the outer mitochondrial membrane, inner mitochondrial membrane mitochondrial and mitochondrial matrix, as demonstrated by assessing the mitochondrial targeting of the following proteins: carnitine palmitoyl transferase 1 (consensus sequence and R123C mutant), acetyl-CoA carboxylase 2, uncoupling protein 1 and holocarboxylase synthetase. Our screen may be useful for linking the mitochondrial proteome with rare diseases and for devising drug- and nutrition-based strategies for altering the mitochondrial targeting of proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

A chemical proteomics approach for global analysis of lysine monomethylome profiling.

PubMed

Wu, Zhixiang; Cheng, Zhongyi; Sun, Mingwei; Wan, Xuelian; Liu, Ping; He, Tieming; Tan, Minjia; Zhao, Yingming

2015-02-01

Methylation of lysine residues on histone proteins is known to play an important role in chromatin structure and function. However, non-histone protein substrates of this modification remain largely unknown. An effective approach for system-wide analysis of protein lysine methylation, particularly lysine monomethylation, is lacking. Here we describe a chemical proteomics approach for global screening for monomethyllysine substrates, involving chemical propionylation of monomethylated lysine, affinity enrichment of the modified monomethylated peptides, and HPLC/MS/MS analysis. Using this approach, we identified with high confidence 446 lysine monomethylation sites in 398 proteins, including three previously unknown histone monomethylation marks, representing the largest data set of protein lysine monomethylation described to date. Our data not only confirms previously discovered lysine methylation substrates in the nucleus and spliceosome, but also reveals new substrates associated with diverse biological processes. This method hence offers a powerful approach for dynamic study of protein lysine monomethylation under diverse cellular conditions and in human diseases. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Isocitrate dehydrogenase mutations confer dasatinib hypersensitivity and SRC-dependence in intrahepatic cholangiocarcinoma

PubMed Central

Saha, Supriya K.; Gordan, John D.; Kleinstiver, Benjamin P.; Vu, Phuong; Najem, Mortada S.; Yeo, Jia-Chi; Shi, Lei; Kato, Yasutaka; Levin, Rebecca S.; Webber, James T.; Damon, Leah J.; Egan, Regina K.; Greninger, Patricia; McDermott, Ultan; Garnett, Mathew J.; Jenkins, Roger L.; Rieger-Christ, Kimberly M.; Sullivan, Travis B.; Hezel, Aram F.; Liss, Andrew S.; Mizukami, Yusuke; Goyal, Lipika; Ferrone, Cristina R.; Zhu, Andrew X.; Joung, J. Keith; Shokat, Kevan M.; Benes, Cyril H.; Bardeesy, Nabeel

2017-01-01

Intrahepatic cholangiocarcinoma (ICC) is an aggressive liver bile duct malignancy exhibiting frequent isocitrate dehydrogenase (IDH1/IDH2) mutations. Through a high-throughput drug screen of a large panel of cancer cell lines including 17 biliary tract cancers, we found that IDH mutant (IDHm) ICC cells demonstrate a striking response to the multi-kinase inhibitor dasatinib, with the highest sensitivity among 682 solid tumor cell lines. Using unbiased proteomics to capture the activated kinome and CRISPR/Cas9-based genome editing to introduce dasatinib-resistant ‘gatekeeper’ mutant kinases, we identified SRC as a critical dasatinib target in IDHm ICC. Importantly, dasatinib-treated IDHm xenografts exhibited pronounced apoptosis and tumor regression. Our results show that IDHm ICC cells have a unique dependency on SRC and suggest that dasatinib may have therapeutic benefit against IDHm ICC. Moreover, these proteomic and genome-editing strategies provide a systematic and broadly applicable approach to define targets of kinase inhibitors underlying drug responsiveness. PMID:27231123

Dried Blood Spot Proteomics: Surface Extraction of Endogenous Proteins Coupled with Automated Sample Preparation and Mass Spectrometry Analysis

NASA Astrophysics Data System (ADS)

Martin, Nicholas J.; Bunch, Josephine; Cooper, Helen J.

2013-08-01

Dried blood spots offer many advantages as a sample format including ease and safety of transport and handling. To date, the majority of mass spectrometry analyses of dried blood spots have focused on small molecules or hemoglobin. However, dried blood spots are a potentially rich source of protein biomarkers, an area that has been overlooked. To address this issue, we have applied an untargeted bottom-up proteomics approach to the analysis of dried blood spots. We present an automated and integrated method for extraction of endogenous proteins from the surface of dried blood spots and sample preparation via trypsin digestion by use of the Advion Biosciences Triversa Nanomate robotic platform. Liquid chromatography tandem mass spectrometry of the resulting digests enabled identification of 120 proteins from a single dried blood spot. The proteins identified cross a concentration range of four orders of magnitude. The method is evaluated and the results discussed in terms of the proteins identified and their potential use as biomarkers in screening programs.

Ribosome surface properties may impose limits on the nature of the cytoplasmic proteome

PubMed Central

2017-01-01

Much of the molecular motion in the cytoplasm is diffusive, which possibly limits the tempo of processes. We studied the dependence of protein mobility on protein surface properties and ionic strength. We used surface-modified fluorescent proteins (FPs) and determined their translational diffusion coefficients (D) in the cytoplasm of Escherichia coli, Lactococcus lactis and Haloferax volcanii. We find that in E. coli D depends on the net charge and its distribution over the protein, with positive proteins diffusing up to 100-fold slower than negative ones. This effect is weaker in L. lactis and Hfx. volcanii due to electrostatic screening. The decrease in mobility is probably caused by interaction of positive FPs with ribosomes as shown in in vivo diffusion measurements and confirmed in vitro with purified ribosomes. Ribosome surface properties may thus limit the composition of the cytoplasmic proteome. This finding lays bare a paradox in the functioning of prokaryotic (endo)symbionts. PMID:29154755

Development and application of a quantitative multiplexed small GTPase activity assay using targeted proteomics.

PubMed

Zhang, Cheng-Cheng; Li, Ru; Jiang, Honghui; Lin, Shujun; Rogalski, Jason C; Liu, Kate; Kast, Juergen

2015-02-06

Small GTPases are a family of key signaling molecules that are ubiquitously expressed in various types of cells. Their activity is often analyzed by western blot, which is limited by its multiplexing capability, the quality of isoform-specific antibodies, and the accuracy of quantification. To overcome these issues, a quantitative multiplexed small GTPase activity assay has been developed. Using four different binding domains, this assay allows the binding of up to 12 active small GTPase isoforms simultaneously in a single experiment. To accurately quantify the closely related small GTPase isoforms, a targeted proteomic approach, i.e., selected/multiple reaction monitoring, was developed, and its functionality and reproducibility were validated. This assay was successfully applied to human platelets and revealed time-resolved coactivation of multiple small GTPase isoforms in response to agonists and differential activation of these isoforms in response to inhibitor treatment. This widely applicable approach can be used for signaling pathway studies and inhibitor screening in many cellular systems.

Tissue matrix arrays for high throughput screening and systems analysis of cell function

PubMed Central

Beachley, Vince Z.; Wolf, Matthew T.; Sadtler, Kaitlyn; Manda, Srikanth S.; Jacobs, Heather; Blatchley, Michael; Bader, Joel S.; Pandey, Akhilesh; Pardoll, Drew; Elisseeff, Jennifer H.

2015-01-01

Cell and protein arrays have demonstrated remarkable utility in the high-throughput evaluation of biological responses; however, they lack the complexity of native tissue and organs. Here, we describe tissue extracellular matrix (ECM) arrays for screening biological outputs and systems analysis. We spotted processed tissue ECM particles as two-dimensional arrays or incorporated them with cells to generate three-dimensional cell-matrix microtissue arrays. We then investigated the response of human stem, cancer, and immune cells to tissue ECM arrays originating from 11 different tissues, and validated the 2D and 3D arrays as representative of the in vivo microenvironment through quantitative analysis of tissue-specific cellular responses, including matrix production, adhesion and proliferation, and morphological changes following culture. The biological outputs correlated with tissue proteomics, and network analysis identified several proteins linked to cell function. Our methodology enables broad screening of ECMs to connect tissue-specific composition with biological activity, providing a new resource for biomaterials research and translation. PMID:26480475

Identification of novel candidate maternal serum protein markers for Down syndrome by integrated proteomic and bioinformatic analysis.

PubMed

Kang, Yuan; Dong, Xinran; Zhou, Qiongjie; Zhang, Ying; Cheng, Yan; Hu, Rong; Su, Cuihong; Jin, Hong; Liu, Xiaohui; Ma, Duan; Tian, Weidong; Li, Xiaotian

2012-03-01

This study aimed to identify candidate protein biomarkers from maternal serum for Down syndrome (DS) by integrated proteomic and bioinformatics analysis. A pregnancy DS group of 18 women and a control group with the same number were prepared, and the maternal serum proteins were analyzed by isobaric tags for relative and absolute quantitation and mass spectrometry, to identify DS differentially expressed maternal serum proteins (DS-DEMSPs). Comprehensive bioinformatics analysis was then employed to analyze DS-DEMSPs both in this paper and seven related publications. Down syndrome differentially expressed maternal serum proteins from different studies are significantly enriched with common Gene Ontology functions, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, transcription factor binding sites, and Pfam protein domains, However, the DS-DEMSPs are less functionally related to known DS-related genes. These evidences suggest that common molecular mechanisms induced by secondary effects may be present upon DS carrying. A simple scoring scheme revealed Alpha-2-macroglobulin, Apolipoprotein A1, Apolipoprotein E, Complement C1s subcomponent, Complement component 5, Complement component 8, alpha polypeptide, Complement component 8, beta polypeptide and Fibronectin as potential DS biomarkers. The integration of proteomics and bioinformatics studies provides a novel approach to develop new prenatal screening methods for noninvasive yet accurate diagnosis of DS. Copyright © 2012 John Wiley & Sons, Ltd.

Nipple aspirate fluid-A liquid biopsy for diagnosing breast health.

PubMed

Shaheed, Sadr-Ul; Tait, Catherine; Kyriacou, Kyriacos; Mullarkey, Joanne; Burrill, Wayne; Patterson, Laurence H; Linforth, Richard; Salhab, Mohamed; Sutton, Chris W

2017-09-01

Nipple secretions are protein-rich and a potential source of breast cancer biomarkers for breast cancer screening. Previous studies of specific proteins have shown limited correlation with clinicopathological features. Our aim, in this pilot study, was to investigate the intra- and interpatient protein composition of nipple secretions and the implications for their use as liquid biopsies. Matched pairs of nipple discharge/nipple aspirate fluid (NAF, n = 15) were characterized for physicochemical properties and SDS-PAGE. Four pairs were selected for semiquantitative proteomic profiling and trypsin-digested peptides analyzed using 2D-LC Orbitrap Fusion MS. The resulting data were subject to bioinformatics analysis and statistical evaluation for functional significance. A total of 1990 unique proteins were identified many of which are established cancer-associated markers. Matched pairs shared the greatest similarity (average Pearson correlation coefficient of 0.94), but significant variations between individuals were observed. This was the most complete proteomic study of nipple discharge/nipple aspirate fluid to date providing a valuable source for biomarker discovery. The high level of milk proteins in healthy volunteer samples compared to the cancer patients was associated with galactorrhoea. Using matched pairs increased confidence in patient-specific protein levels but changes relating to cancer stage require investigation of a larger cohort. © 2017 The Authors. PROTEOMICS-Clinical Applications published by WILEY-VCH Verlag GmbH & Co. KGaA.

Identification of the pI 4.6 extensin peroxidase from Lycopersicon esculentum using proteomics and reverse-genomics.

PubMed

Dong, Wen; Kieliszewski, Marcia; Held, Michael A

2015-04-01

The regulation of plant cell growth and early defense response involves the insolubilization of hydroxyproline-rich glycoproteins (HRGPs), such as extensin, in the primary cell wall. In tomato (Lycopersicon esculentum), insolubilization occurs by the formation of tyrosyl-crosslinks catalyzed specifically by the pI 4.6 extensin peroxidase (EP). To date, neither the gene encoding EP nor the protein itself has been identified. Here, we have identified tomato EP candidates using both proteomic and bioinformatic approaches. Bioinformatic screening of the tomato genome yielded eight EP candidates, which contained a putative signal sequence and a predicted pI near 4.6. Biochemical fractionation of tomato culture media followed by proteomic detection further refined our list of EP candidates to three, with the lead candidate designated (CG5). To test for EP crosslinking activity, we cloned into a bacterial expression vector the CG5 open-reading frame from tomato cDNA. The CG5 was expressed in Escherichia coli, fractionated from inclusion bodies, and folded in vitro. The peroxidase activity of CG5 was assayed and quantified by ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) assay. Subsequent extensin crosslinking assays showed that CG5 can covalently crosslink authentic tomato P1 extensin and P3-type extensin analogs in vitro supporting our hypothesis that CG5 encodes a tomato EP. Copyright © 2014 Elsevier Ltd. All rights reserved.

Identification of the pI 4.6 extensin peroxidase from Lycopersicon esculentum using proteomics and reverse-genomics

PubMed Central

Dong, Wen; Kieliszewski, Marcia; Held, Michael A.

2014-01-01

The regulation of plant cell growth and early defense response involves the insolubilization of hydroxyproline-rich glycoproteins (HRGPs), such as extensin, in the primary cell wall. In tomato (Lycopersicon esculentum), insolublization occurs by the formation of tyrosyl-crosslinks catalyzed specifically by the pI 4.6 extensin peroxidase (EP). To date, neither the gene encoding EP nor the protein itself has been identified. Here, we’ve identified tomato EP candidates using both proteomic and bioinformatic approaches. Bioinformatic screening of the tomato genome yielded eight EP candidates, which contained a putative signal sequence and a predicted pI near 4.6. Biochemical fractionation of tomato culture media followed by proteomic detection further refined our list of EP candidates to three, with the lead candidate designated (CG5). To test for EP crosslinking activity, we cloned into a bacterial expression vector the CG5 open-reading frame from tomato cDNA. The CG5 was expressed in E. coli, fractionated from inclusion bodies, and folded in vitro. The peroxidase activity of CG5 was assayed and quantified by ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) assay. Subsequent extensin crosslinking assays showed that CG5 can covalently crosslink authentic tomato P1 extensin and P3-type extensin analogs in vitro supporting our hypothesis that CG5 encodes a tomato EP. PMID:25446231

Efficient farnesylation of an extended C-terminal C(x)3X sequence motif expands the scope of the prenylated proteome.

PubMed

Blanden, Melanie J; Suazo, Kiall F; Hildebrandt, Emily R; Hardgrove, Daniel S; Patel, Meet; Saunders, William P; Distefano, Mark D; Schmidt, Walter K; Hougland, James L

2018-02-23

Protein prenylation is a post-translational modification that has been most commonly associated with enabling protein trafficking to and interaction with cellular membranes. In this process, an isoprenoid group is attached to a cysteine near the C terminus of a substrate protein by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I or II (GGTase-I and GGTase-II). FTase and GGTase-I have long been proposed to specifically recognize a four-amino acid C AAX C-terminal sequence within their substrates. Surprisingly, genetic screening reveals that yeast FTase can modify sequences longer than the canonical C AAX sequence, specifically C( x ) 3 X sequences with four amino acids downstream of the cysteine. Biochemical and cell-based studies using both peptide and protein substrates reveal that mammalian FTase orthologs can also prenylate C( x ) 3 X sequences. As the search to identify physiologically relevant C( x ) 3 X proteins begins, this new prenylation motif nearly doubles the number of proteins within the yeast and human proteomes that can be explored as potential FTase substrates. This work expands our understanding of prenylation's impact within the proteome, establishes the biologically relevant reactivity possible with this new motif, and opens new frontiers in determining the impact of non-canonically prenylated proteins on cell function. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Integrated proteomics, genomics, metabolomics approaches reveal oxalic acid as pathogenicity factor in Tilletia indica inciting Karnal bunt disease of wheat.

PubMed

Pandey, Vishakha; Singh, Manoj; Pandey, Dinesh; Kumar, Anil

2018-05-18

Tilletia indica incites Karnal bunt (KB) disease in wheat. To date, no KB resistant wheat cultivar could be developed due to non-availability of potential biomarkers related to pathogenicity/virulence for screening of resistant wheat genotypes. The present study was carried out to compare the proteomes of T. indica highly (TiK) and low (TiP) virulent isolates. Twenty one protein spots consistently observed as up-regulated/differential in the TiK proteome were selected for identification by MALDI-TOF/TOF. Identified sequences showed homology with fungal proteins playing essential role in plant infection and pathogen survival, including stress response, adhesion, fungal penetration, invasion, colonization, degradation of host cell wall, signal transduction pathway. These results were integrated with T. indica genome sequence for identification of homologs of candidate pathogenicity/virulence related proteins. Protein identified in TiK isolate as malate dehydrogenase that converts malate to oxaloacetate which is precursor of oxalic acid. Oxalic acid is key pathogenicity factor in phytopathogenic fungi. These results were validated by GC-MS based metabolic profiling of T. indica isolates indicating that oxalic acid was exclusively identified in TiK isolate. Thus, integrated omics approaches leads to identification of pathogenicity/virulence factor(s) that would provide insights into pathogenic mechanisms of fungi and aid in devising effective disease management strategies.

Two-dimensional electrophoretic profiling of normal human kidney glomerulus proteome and construction of an extensible markup language (XML)-based database.

PubMed

Yoshida, Yutaka; Miyazaki, Kenji; Kamiie, Junichi; Sato, Masao; Okuizumi, Seiji; Kenmochi, Akihisa; Kamijo, Ken'ichi; Nabetani, Takuji; Tsugita, Akira; Xu, Bo; Zhang, Ying; Yaoita, Eishin; Osawa, Tetsuo; Yamamoto, Tadashi

2005-03-01

To contribute to physiology and pathophysiology of the glomerulus of human kidney, we have launched a proteomic study of human glomerulus, and compiled a profile of proteins expressed in the glomerulus of normal human kidney by two-dimensional gel electrophoresis (2-DE) and identification with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and/or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Kidney cortices with normal appearance were obtained from patients under surgical nephrectomy due to renal tumor, and glomeruli were highly purified by a standard sieving method followed by picking-up under a phase-contrast microscope. The glomerular proteins were separated by 2-DE with 24 cm immobilized pH gradient strips in the 3-10 range in the first dimension and 26 x 20 cm sodium dodecyl sulfate polyacrylamide electrophoresis gels of 12.5% in the second dimension. Gels were silver-stained, and valid spots were processed for identification through an integrated robotic system that consisted of a spot picker, an in-gel digester, and a MALDI-TOF MS and / or a LC-MS/MS. From 2-DE gel images of glomeruli of four subjects with no apparent pathologic manifestations, a synthetic gel image of normal glomerular proteins was created. The synthetic gel image contained 1713 valid spots, of which 1559 spots were commonly observed in the respective 2-DE gels. Among the 1559 spots, 347 protein spots, representing 212 proteins, have so far been identified, and used for the construction of an extensible markup language (XML)-based database. The database is deposited on a web site (http://www.sw.nec.co.jp/bio/rd/hgldb/index.html) in a form accessible to researchers to contribute to proteomic studies of human glomerulus in health and disease.

Studies of the expression of human poly(ADP-ribose) polymerase-1 in Saccharomyces cerevisiae and identification of PARP-1 substrates by yeast proteome microarray screening.

PubMed

Tao, Zhihua; Gao, Peng; Liu, Hung-Wen

2009-12-15

Poly(ADP-ribosyl)ation of various nuclear proteins catalyzed by a family of NAD(+)-dependent enzymes, poly(ADP-ribose) polymerases (PARPs), is an important posttranslational modification reaction. PARP activity has been demonstrated in all types of eukaryotic cells with the exception of yeast, in which the expression of human PARP-1 was shown to lead to retarded cell growth. We investigated the yeast growth inhibition caused by human PARP-1 expression in Saccharomyces cerevisiae. Flow cytometry analysis reveals that PARP-1-expressing yeast cells accumulate in the G(2)/M stage of the cell cycle. Confocal microscopy analysis shows that human PARP-1 is distributed throughout the nucleus of yeast cells but is enriched in the nucleolus. Utilizing yeast proteome microarray screening, we identified 33 putative PARP-1 substrates, six of which are known to be involved in ribosome biogenesis. The poly(ADP-ribosyl)ation of three of these yeast proteins, together with two human homologues, was confirmed by an in vitro PARP-1 assay. Finally, a polysome profile analysis using sucrose gradient ultracentrifugation demonstrated that the ribosome levels in yeast cells expressing PARP-1 are lower than those in control yeast cells. Overall, our data suggest that human PARP-1 may affect ribosome biogenesis by modifying certain nucleolar proteins in yeast. The artificial PARP-1 pathway in yeast may be used as a simple platform to identify substrates and verify function of this important enzyme.

Proteomic Screening and Lasso Regression Reveal Differential Signaling in Insulin and Insulin-like Growth Factor I (IGF1) Pathways.

PubMed

Erdem, Cemal; Nagle, Alison M; Casa, Angelo J; Litzenburger, Beate C; Wang, Yu-Fen; Taylor, D Lansing; Lee, Adrian V; Lezon, Timothy R

2016-09-01

Insulin and insulin-like growth factor I (IGF1) influence cancer risk and progression through poorly understood mechanisms. To better understand the roles of insulin and IGF1 signaling in breast cancer, we combined proteomic screening with computational network inference to uncover differences in IGF1 and insulin induced signaling. Using reverse phase protein array, we measured the levels of 134 proteins in 21 breast cancer cell lines stimulated with IGF1 or insulin for up to 48 h. We then constructed directed protein expression networks using three separate methods: (i) lasso regression, (ii) conventional matrix inversion, and (iii) entropy maximization. These networks, named here as the time translation models, were analyzed and the inferred interactions were ranked by differential magnitude to identify pathway differences. The two top candidates, chosen for experimental validation, were shown to regulate IGF1/insulin induced phosphorylation events. First, acetyl-CoA carboxylase (ACC) knock-down was shown to increase the level of mitogen-activated protein kinase (MAPK) phosphorylation. Second, stable knock-down of E-Cadherin increased the phospho-Akt protein levels. Both of the knock-down perturbations incurred phosphorylation responses stronger in IGF1 stimulated cells compared with insulin. Overall, the time-translation modeling coupled to wet-lab experiments has proven to be powerful in inferring differential interactions downstream of IGF1 and insulin signaling, in vitro. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The hydroxyl-functionalized magnetic particles for purification of glycan-binding proteins.

PubMed

Sun, Xiuxuan; Yang, Ganglong; Sun, Shisheng; Quan, Rui; Dai, Weiwei; Li, Bin; Chen, Chao; Li, Zheng

2009-12-01

Glycan-protein interactions play important biological roles in biological processes. Although there are some methods such as glycan arrays that may elucidate recognition events between carbohydrates and protein as well as screen the important glycan-binding proteins, there is a lack of simple effectively separate method to purify them from complex samples. In proteomics studies, fractionation of samples can help to reduce their complexity and to enrich specific classes of proteins for subsequent downstream analyses. Herein, a rapid simple method for purification of glycan-binding proteins from proteomic samples was developed using hydroxyl-coated magnetic particles coupled with underivatized carbohydrate. Firstly, the epoxy-coated magnetic particles were further hydroxyl functionalized with 4-hydroxybenzhydrazide, then the carbohydrates were efficiently immobilized on hydroxyl functionalized surface of magnetic particles by formation of glycosidic bond with the hemiacetal group at the reducing end of the suitable carbohydrates via condensation. All conditions of this method were optimized. The magnetic particle-carbohydrate conjugates were used to purify the glycan-binding proteins from human serum. The fractionated glycan-binding protein population was displayed by SDS-PAGE. The result showed that the amount of 1 mg magnetic particles coupled with mannose in acetate buffer (pH 5.4) was 10 micromol. The fractionated glycan-binding protein population in human serum could be eluted from the magnetic particle-mannose conjugates by 0.1% SDS. The methodology could work together with the glycan microarrays for screening and purification of the important GBPs from complex protein samples.

SWI/SNF Chromatin-remodeling Factors: Multiscale Analyses and Diverse Functions*

PubMed Central

Euskirchen, Ghia; Auerbach, Raymond K.; Snyder, Michael

2012-01-01

Chromatin-remodeling enzymes play essential roles in many biological processes, including gene expression, DNA replication and repair, and cell division. Although one such complex, SWI/SNF, has been extensively studied, new discoveries are still being made. Here, we review SWI/SNF biochemistry; highlight recent genomic and proteomic advances; and address the role of SWI/SNF in human diseases, including cancer and viral infections. These studies have greatly increased our understanding of complex nuclear processes. PMID:22952240

Morphologic and proteomic characterization of exosomes released by cultured extravillous trophoblast cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Atay, Safinur; Gercel-Taylor, Cicek; Kesimer, Mehmet

Exosomes represent an important intercellular communication vehicle, mediating events essential for the decidual microenvironment. While we have demonstrated exosome induction of pro-inflammatory cytokines, to date, no extensive characterization of trophoblast-derived exosomes has been provided. Our objective was to provide a morphologic and proteomic characterization of these exosomes. Exosomes were isolated from the conditioned media of Swan71 human trophoblast cells by ultrafiltration and ultracentrifugation. These were analyzed for density (sucrose density gradient centrifugation), morphology (electron microscopy), size (dynamic light scattering) and protein composition (Ion Trap mass spectrometry and western immunoblotting). Based on density gradient centrifugation, microvesicles from Sw71 cells exhibit amore » density between 1.134 and 1.173 g/ml. Electron microscopy demonstrated that microvesicles from Sw71 cells exhibit the characteristic cup-shaped morphology of exosomes. Dynamic light scattering showed a bell-shaped curve, indicating a homogeneous population with a mean size of 165 nm {+-} 0.5 nm. Ion Trap mass spectrometry demonstrated the presence of exosome marker proteins (including CD81, Alix, cytoskeleton related proteins, and Rab family). The MS results were confirmed by western immunoblotting. Based on morphology, density, size and protein composition, we defined the release of exosomes from extravillous trophoblast cells and provide their first extensive characterization. This characterization is essential in furthering our understanding of 'normal' early pregnancy.« less

Expansion of 3D human induced pluripotent stem cell aggregates in bioreactors: Bioprocess intensification and scaling-up approaches.

PubMed

Abecasis, Bernardo; Aguiar, Tiago; Arnault, Émilie; Costa, Rita; Gomes-Alves, Patricia; Aspegren, Anders; Serra, Margarida; Alves, Paula M

2017-03-20

Human induced pluripotent stem cells (hiPSC) are attractive tools for drug screening and disease modeling and promising candidates for cell therapy applications. However, to achieve the high numbers of cells required for these purposes, scalable and clinical-grade technologies must be established. In this study, we use environmentally controlled stirred-tank bioreactors operating in perfusion as a powerful tool for bioprocess intensification of hiPSC production. We demonstrate the importance of controlling the dissolved oxygen concentration at low levels (4%) and perfusion at 1.3day -1 dilution rate to improve hiPSC growth as aggregates in a xeno-free medium. This strategy allowed for increased cell specific growth rate, maximum volumetric concentrations (4.7×10 6 cell/mL) and expansion factors (approximately 19 in total cells), resulting in a 2.6-fold overall improvement in cell yields. Extensive cell characterization, including whole proteomic analysis, was performed to confirm that cells' pluripotent phenotype was maintained during culture. A scalable protocol for continuous expansion of hiPSC aggregates in bioreactors was implemented using mechanical dissociation for aggregate disruption and cell passaging. A total expansion factor of 1100 in viable cells was obtained in 11days of culture, while cells maintained their proliferation capacity, pluripotent phenotype and potential as well as genomic stability after 3 sequential passages in bioreactors. Copyright © 2017 Elsevier B.V. All rights reserved.

Designed protein reveals structural determinants of extreme kinetic stability

PubMed Central

Broom, Aron; Ma, S. Martha; Xia, Ke; Rafalia, Hitesh; Trainor, Kyle; Colón, Wilfredo; Gosavi, Shachi; Meiering, Elizabeth M.

2015-01-01

The design of stable, functional proteins is difficult. Improved design requires a deeper knowledge of the molecular basis for design outcomes and properties. We previously used a bioinformatics and energy function method to design a symmetric superfold protein composed of repeating structural elements with multivalent carbohydrate-binding function, called ThreeFoil. This and similar methods have produced a notably high yield of stable proteins. Using a battery of experimental and computational analyses we show that despite its small size and lack of disulfide bonds, ThreeFoil has remarkably high kinetic stability and its folding is specifically chaperoned by carbohydrate binding. It is also extremely stable against thermal and chemical denaturation and proteolytic degradation. We demonstrate that the kinetic stability can be predicted and modeled using absolute contact order (ACO) and long-range order (LRO), as well as coarse-grained simulations; the stability arises from a topology that includes many long-range contacts which create a large and highly cooperative energy barrier for unfolding and folding. Extensive data from proteomic screens and other experiments reveal that a high ACO/LRO is a general feature of proteins with strong resistances to denaturation and degradation. These results provide tractable approaches for predicting resistance and designing proteins with sufficient topological complexity and long-range interactions to accommodate destabilizing functional features as well as withstand chemical and proteolytic challenge. PMID:26554002

Genomics of Dementia: APOE- and CYP2D6-Related Pharmacogenetics

PubMed Central

Cacabelos, Ramón; Martínez, Rocío; Fernández-Novoa, Lucía; Carril, Juan C.; Lombardi, Valter; Carrera, Iván; Corzo, Lola; Tellado, Iván; Leszek, Jerzy; McKay, Adam; Takeda, Masatoshi

2012-01-01

Dementia is a major problem of health in developed societies. Alzheimer's disease (AD), vascular dementia, and mixed dementia account for over 90% of the most prevalent forms of dementia. Both genetic and environmental factors are determinant for the phenotypic expression of dementia. AD is a complex disorder in which many different gene clusters may be involved. Most genes screened to date belong to different proteomic and metabolomic pathways potentially affecting AD pathogenesis. The ε4 variant of the APOE gene seems to be a major risk factor for both degenerative and vascular dementia. Metabolic factors, cerebrovascular disorders, and epigenetic phenomena also contribute to neurodegeneration. Five categories of genes are mainly involved in pharmacogenomics: genes associated with disease pathogenesis, genes associated with the mechanism of action of a particular drug, genes associated with phase I and phase II metabolic reactions, genes associated with transporters, and pleiotropic genes and/or genes associated with concomitant pathologies. The APOE and CYP2D6 genes have been extensively studied in AD. The therapeutic response to conventional drugs in patients with AD is genotype specific, with CYP2D6-PMs, CYP2D6-UMs, and APOE-4/4 carriers acting as the worst responders. APOE and CYP2D6 may cooperate, as pleiotropic genes, in the metabolism of drugs and hepatic function. The introduction of pharmacogenetic procedures into AD pharmacological treatment may help to optimize therapeutics. PMID:22482072

A comprehensive evaluation of popular proteomics software workflows for label-free proteome quantification and imputation.

PubMed

Välikangas, Tommi; Suomi, Tomi; Elo, Laura L

2017-05-31

Label-free mass spectrometry (MS) has developed into an important tool applied in various fields of biological and life sciences. Several software exist to process the raw MS data into quantified protein abundances, including open source and commercial solutions. Each software includes a set of unique algorithms for different tasks of the MS data processing workflow. While many of these algorithms have been compared separately, a thorough and systematic evaluation of their overall performance is missing. Moreover, systematic information is lacking about the amount of missing values produced by the different proteomics software and the capabilities of different data imputation methods to account for them.In this study, we evaluated the performance of five popular quantitative label-free proteomics software workflows using four different spike-in data sets. Our extensive testing included the number of proteins quantified and the number of missing values produced by each workflow, the accuracy of detecting differential expression and logarithmic fold change and the effect of different imputation and filtering methods on the differential expression results. We found that the Progenesis software performed consistently well in the differential expression analysis and produced few missing values. The missing values produced by the other software decreased their performance, but this difference could be mitigated using proper data filtering or imputation methods. Among the imputation methods, we found that the local least squares (lls) regression imputation consistently increased the performance of the software in the differential expression analysis, and a combination of both data filtering and local least squares imputation increased performance the most in the tested data sets. © The Author 2017. Published by Oxford University Press.

Quantitative multiplexed proteomics of Taenia solium cysts obtained from the skeletal muscle and central nervous system of pigs

PubMed Central

Navarrete-Perea, José; Isasa, Marta; Paulo, Joao A.; Corral-Corral, Ricardo; Flores-Bautista, Jeanette; Hernández-Téllez, Beatriz; Bobes, Raúl J.; Fragoso, Gladis; Sciutto, Edda; Soberón, Xavier; Gygi, Steven P.; Laclette, Juan P.

2017-01-01

In human and porcine cysticercosis caused by the tapeworm Taenia solium, the larval stage (cysts) can infest several tissues including the central nervous system (CNS) and the skeletal muscles (SM). The cyst’s proteomics changes associated with the tissue localization in the host tissues have been poorly studied. Quantitative multiplexed proteomics has the power to evaluate global proteome changes in response to different conditions. Here, using a TMT-multiplexed strategy we identified and quantified over 4,200 proteins in cysts obtained from the SM and CNS of pigs, of which 891 were host proteins. To our knowledge, this is the most extensive intermixing of host and parasite proteins reported for tapeworm infections.Several antigens in cysticercosis, i.e., GP50, paramyosin and a calcium-binding protein were enriched in skeletal muscle cysts. Our results suggested the occurrence of tissue-enriched antigen that could be useful in the improvement of the immunodiagnosis for cysticercosis. Using several algorithms for epitope detection, we selected 42 highly antigenic proteins enriched for each tissue localization of the cysts. Taking into account the fold changes and the antigen/epitope contents, we selected 10 proteins and produced synthetic peptides from the best epitopes. Nine peptides were recognized by serum antibodies of cysticercotic pigs, suggesting that those peptides are antigens. Mixtures of peptides derived from SM and CNS cysts yielded better results than mixtures of peptides derived from a single tissue location, however the identification of the ‘optimal’ tissue-enriched antigens remains to be discovered. Through machine learning technologies, we determined that a reliable immunodiagnostic test for porcine cysticercosis required at least five different antigenic determinants. PMID:28945737

Metabolic Reprogramming During Purine Stress in the Protozoan Pathogen Leishmania donovani

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martin, Jessica L.; Yates, Phillip A.; Soysa, Radika

The ability of Leishmania to survive in their insect or mammalian host is dependent upon an ability to sense and adapt to changes in the microenvironment. However, little is known about the molecular mechanisms underlying the parasite response to environmental changes, such as nutrient availability. To elucidate nutrient stress response pathways in Leishmania donovani, we have used purine starvation as the paradigm. The salvage of purines from the host milieu is obligatory for parasite replication; nevertheless, purine-starved parasites can persist in culture without supplementary purine for over 3 months, indicating that the response to purine starvation is robust and engendersmore » parasite survival under conditions of extreme scarcity. To understand metabolic reprogramming during purine starvation we have employed global approaches. Whole proteome comparisons between purine-starved and purine-replete parasites over a 6-48 h span have revealed a temporal and coordinated response to purine starvation. Purine transporters and enzymes involved in acquisition at the cell surface are upregulated within a few hours of purine removal from the media, while other key purine salvage components are upregulated later in the time-course and more modestly. After 48 h, the proteome of purine-starved parasites is extensively remodeled and adaptations to purine stress appear tailored to deal with both purine deprivation and general stress. To probe the molecular mechanisms affecting proteome remodeling in response to purine starvation, comparative RNA-seq analyses, qRT-PCR, and luciferase reporter assays were performed on purine-starved versus purine-replete parasites. While the regulation of a minority of proteins tracked with changes at the mRNA level, for many regulated proteins it appears that proteome remodeling during purine stress occurs primarily via translational and/or post-translational mechanisms.« less

Characterization and Comprehensive Proteome Profiling of Exosomes Secreted by Hepatocytes

PubMed Central

Conde-Vancells, Javier; Rodriguez-Suarez, Eva; Embade, Nieves; Gil, David; Matthiesen, Rune; Valle, Mikel; Elortza, Felix; Lu, Shelly C.; Mato, Jose M.; Falcon-Perez, Juan M.

2009-01-01

Synopsis Exosomes constitute a discrete population of nanometer-sized (30-150 nm) vesicles formed in endocytic compartments and released to the extracellular environment by different cell types. In this work we demonstrated by electron microscopic, western blotting and proteomic analyses that primary hepatocytes secrete exosome-like vesicles containing proteins involved in metabolizing lipoproteins, endogenous compounds as well as xenobiotics. These new findings contribute to improve our knowledge about biology's hepatocyte and may have important diagnostic, prognosis and therapeutic implications in liver diseases Exosomes represent a discrete population of vesicles that are secreted from various cell types to the extracellular media. Their protein and lipid composition are a consequence of sorting events at the level of the multivesicular body, a central organelle which integrates endocytic and secretory pathways. Characterization of exosomes from different biological samples has shown the presence of common as well as cell-type specific proteins. Remarkably, the protein content of the exosomes is modified upon pathological or stress conditions. Hepatocytes play a central role in the body response to stress metabolizing potentially harmful endogenous substances as well as xenobiotics. In the present study we described and characterized for first time exosome secretion in non-tumoral hepatocytes, and using a systematic proteomic approach, we establish the first extensive proteome of a hepatocyte-derived exosome population which should be useful in furthering our understanding of the hepatic function and in the identification of components that may serve as biomarkers for hepatic alterations. Our analysis identifies a significant number of proteins previously described among exosomes derived from others cell types as well as proteins involved in metabolizing lipoproteins, endogenous compounds and xenobiotics, not previously described in exosomes. Furthermore, we demonstrated that exosomal membrane proteins can constitute an interesting tool to express non-exosomal proteins into exosomes with therapeutic purposes. PMID:19367702

Characterization of the Proteome of Theobroma cacao Beans by Nano-UHPLC-ESI MS/MS.

PubMed

Scollo, Emanuele; Neville, David; Oruna-Concha, M Jose; Trotin, Martine; Cramer, Rainer

2018-02-01

Cocoa seed storage proteins play an important role in flavour development as aroma precursors are formed from their degradation during fermentation. Major proteins in the beans of Theobroma cacao are the storage proteins belonging to the vicilin and albumin classes. Although both these classes of proteins have been extensively characterized, there is still limited information on the expression and abundance of other proteins present in cocoa beans. This work is the first attempt to characterize the whole cocoa bean proteome by nano-UHPLC-ESI MS/MS analysis using tryptic digests of cocoa bean protein extracts. The results of this analysis show that >1000 proteins could be identified using a species-specific Theobroma cacao database. The majority of the identified proteins were involved with metabolism and energy. Additionally, a significant number of the identified proteins were linked to protein synthesis and processing. Several proteins were also involved with plant response to stress conditions and defence. Albumin and vicilin storage proteins showed the highest intensity values among all detected proteins, although only seven entries were identified as storage proteins. A comparison of MS/MS data searches carried out against larger non-specific databases confirmed that using a species-specific database can increase the number of identified proteins, and at the same time reduce the number of false positives. The results of this work will be useful in developing tools that can allow the comparison of the proteomic profile of cocoa beans from different genotypes and geographic origins. Data are available via ProteomeXchange with identifier PXD005586. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Serum biomarkers of Burkholderia mallei infection elucidated by proteomic imaging of skin and lung abscesses.

PubMed

Glaros, Trevor G; Blancett, Candace D; Bell, Todd M; Natesan, Mohan; Ulrich, Robert G

2015-01-01

The bacterium Burkholderia mallei is the etiological agent of glanders, a highly contagious, often fatal zoonotic infectious disease that is also a biodefense concern. Clinical laboratory assays that analyze blood or other biological fluids are the highest priority because these specimens can be collected with minimal risk to the patient. However, progress in developing sensitive assays for monitoring B. mallei infection is hampered by a shortage of useful biomarkers. Reasoning that there should be a strong correlation between the proteomes of infected tissues and circulating serum, we employed imaging mass spectrometry (IMS) of thin-sectioned tissues from Chlorocebus aethiops (African green) monkeys infected with B. mallei to localize host and pathogen proteins that were associated with abscesses. Using laser-capture microdissection of specific regions identified by IMS and histology within the tissue sections, a more extensive proteomic analysis was performed by a technique that combined the physical separation capabilities of liquid chromatography (LC) with the sensitive mass analysis capabilities of mass spectrometry (LC-MS/MS). By examining standard formalin-fixed, paraffin-embedded tissue sections, this strategy resulted in the identification of several proteins that were associated with lung and skin abscesses, including the host protein calprotectin and the pathogen protein GroEL. Elevated levels of calprotectin detected by ELISA and antibody responses to GroEL, measured by a microarray of the bacterial proteome, were subsequently detected in the sera of C. aethiops, Macaca mulatta, and Macaca fascicularis primates infected with B. mallei. Our results demonstrate that a combination of multidimensional MS analysis of traditional histology specimens with high-content protein microarrays can be used to discover lead pairs of host-pathogen biomarkers of infection that are identifiable in biological fluids.

Quantitative multiplexed proteomics of Taenia solium cysts obtained from the skeletal muscle and central nervous system of pigs.

PubMed

Navarrete-Perea, José; Isasa, Marta; Paulo, Joao A; Corral-Corral, Ricardo; Flores-Bautista, Jeanette; Hernández-Téllez, Beatriz; Bobes, Raúl J; Fragoso, Gladis; Sciutto, Edda; Soberón, Xavier; Gygi, Steven P; Laclette, Juan P

2017-09-01

In human and porcine cysticercosis caused by the tapeworm Taenia solium, the larval stage (cysts) can infest several tissues including the central nervous system (CNS) and the skeletal muscles (SM). The cyst's proteomics changes associated with the tissue localization in the host tissues have been poorly studied. Quantitative multiplexed proteomics has the power to evaluate global proteome changes in response to different conditions. Here, using a TMT-multiplexed strategy we identified and quantified over 4,200 proteins in cysts obtained from the SM and CNS of pigs, of which 891 were host proteins. To our knowledge, this is the most extensive intermixing of host and parasite proteins reported for tapeworm infections.Several antigens in cysticercosis, i.e., GP50, paramyosin and a calcium-binding protein were enriched in skeletal muscle cysts. Our results suggested the occurrence of tissue-enriched antigen that could be useful in the improvement of the immunodiagnosis for cysticercosis. Using several algorithms for epitope detection, we selected 42 highly antigenic proteins enriched for each tissue localization of the cysts. Taking into account the fold changes and the antigen/epitope contents, we selected 10 proteins and produced synthetic peptides from the best epitopes. Nine peptides were recognized by serum antibodies of cysticercotic pigs, suggesting that those peptides are antigens. Mixtures of peptides derived from SM and CNS cysts yielded better results than mixtures of peptides derived from a single tissue location, however the identification of the 'optimal' tissue-enriched antigens remains to be discovered. Through machine learning technologies, we determined that a reliable immunodiagnostic test for porcine cysticercosis required at least five different antigenic determinants.

Quantitative proteomic analysis of the brainstem following lethal sarin exposure.

PubMed

Meade, Mitchell L; Hoffmann, Andrea; Makley, Meghan K; Snider, Thomas H; Schlager, John J; Gearhart, Jeffery M

2015-06-22

The brainstem represents a major tissue area affected by sarin organophosphate poisoning due to its function in respiratory and cardiovascular control. While the acute toxic effects of sarin on brainstem-related responses are relatively unknown, other brain areas e.g., cortex or cerebellum, have been studied more extensively. The study objective was to analyze the guinea pig brainstem toxicology response following sarin (2×LD50) exposure by proteome pathway analysis to gain insight into the complex regulatory mechanisms that lead to impairment of respiratory and cardiovascular control. Guinea pig exposure to sarin resulted in the typical acute behavior/physiology outcomes with death between 15 and 25min. In addition, brain and blood acetylcholinesterase activity was significantly reduced in the presence of sarin to 95%, and 89%, respectively, of control values. Isobaric-tagged (iTRAQ) liquid chromatography tandem mass spectrometry (LC-MS/MS) identified 198 total proteins of which 23% were upregulated, and 18% were downregulated following sarin exposure. Direct gene ontology (GO) analysis revealed a sarin-specific broad-spectrum proteomic profile including glutamate-mediated excitotoxicity, calcium overload, energy depletion responses, and compensatory carbohydrate metabolism, increases in ROS defense, DNA damage and chromatin remodeling, HSP response, targeted protein degradation (ubiquitination) and cell death response. With regards to the sarin-dependent effect on respiration, our study supports the potential interference of sarin with CO2/H(+) sensitive chemoreceptor neurons of the brainstem retrotrapezoid nucleus (RTN) that send excitatory glutamergic projections to the respiratory centers. In conclusion, this study gives insight into the brainstem broad-spectrum proteome following acute sarin exposure and the gained information will assist in the development of novel countermeasures. Published by Elsevier B.V.

Regional Screening Levels (RSLs) - User's Guide

EPA Pesticide Factsheets

Regional Screening Level RSL user guide provides extensive guidance for risk assessors doing Chemical Risk Assessment preliminary remediation goal PRG risk based concentration RBC and rick calculations for the assess

Poly-lactic acid nanoparticles (PLA-NP) promote physiological modifications in lung epithelial cells and are internalized by clathrin-coated pits and lipid rafts.

PubMed

da Luz, Camila Macedo; Boyles, Matthew Samuel Powys; Falagan-Lotsch, Priscila; Pereira, Mariana Rodrigues; Tutumi, Henrique Rudolf; de Oliveira Santos, Eidy; Martins, Nathalia Balthazar; Himly, Martin; Sommer, Aniela; Foissner, Ilse; Duschl, Albert; Granjeiro, José Mauro; Leite, Paulo Emílio Corrêa

2017-01-31

Poly-lactic acid nanoparticles (PLA-NP) are a type of polymeric NP, frequently used as nanomedicines, which have advantages over metallic NP such as the ability to maintain therapeutic drug levels for sustained periods of time. Despite PLA-NP being considered biocompatible, data concerning alterations in cellular physiology are scarce. We conducted an extensive evaluation of PLA-NP biocompatibility in human lung epithelial A549 cells using high throughput screening and more complex methodologies. These included measurements of cytotoxicity, cell viability, immunomodulatory potential, and effects upon the cells' proteome. We used non- and green-fluorescent PLA-NP with 63 and 66 nm diameters, respectively. Cells were exposed with concentrations of 2, 20, 100 and 200 µg/mL, for 24, 48 and 72 h, in most experiments. Moreover, possible endocytic mechanisms of internalization of PLA-NP were investigated, such as those involving caveolae, lipid rafts, macropinocytosis and clathrin-coated pits. Cell viability and proliferation were not altered in response to PLA-NP. Multiplex analysis of secreted mediators revealed a low-level reduction of IL-12p70 and vascular epidermal growth factor (VEGF) in response to PLA-NP, while all other mediators assessed were unaffected. However, changes to the cells' proteome were observed in response to PLA-NP, and, additionally, the cellular stress marker miR155 was found to reduce. In dual exposures of staurosporine (STS) with PLA-NP, PLA-NP enhanced susceptibility to STS-induced cell death. Finally, PLA-NP were rapidly internalized in association with clathrin-coated pits, and, to a lesser extent, with lipid rafts. These data demonstrate that PLA-NP are internalized and, in general, tolerated by A549 cells, with no cytotoxicity and no secretion of pro-inflammatory mediators. However, PLA-NP exposure may induce modification of biological functions of A549 cells, which should be considered when designing drug delivery systems. Moreover, the pathways of PLA-NP internalization we detected could contribute to the improvement of selective uptake strategies.

Comprehensive proteome analysis of Actinoplanes sp. SE50/110 highlighting the location of proteins encoded by the acarbose and the pyochelin biosynthesis gene cluster.

PubMed

Wendler, Sergej; Otto, Andreas; Ortseifen, Vera; Bonn, Florian; Neshat, Armin; Schneiker-Bekel, Susanne; Walter, Frederik; Wolf, Timo; Zemke, Till; Wehmeier, Udo F; Hecker, Michael; Kalinowski, Jörn; Becher, Dörte; Pühler, Alfred

2015-07-01

Acarbose is an α-glucosidase inhibitor produced by Actinoplanes sp. SE50/110 that is medically important due to its application in the treatment of type2 diabetes. In this work, a comprehensive proteome analysis of Actinoplanes sp. SE50/110 was carried out to determine the location of proteins of the acarbose (acb) and the putative pyochelin (pch) biosynthesis gene cluster. Therefore, a comprehensive state-of-the-art proteomics approach combining subcellular fractionation, shotgun proteomics and spectral counting to assess the relative abundance of proteins within fractions was applied. The analysis of four different proteome fractions (cytosolic, enriched membrane, membrane shaving and extracellular fraction) resulted in the identification of 1582 of the 8270 predicted proteins. All 22 Acb-proteins and 21 of the 23 Pch-proteins were detected. Predicted membrane-associated, integral membrane or extracellular proteins of the pch and the acb gene cluster were found among the most abundant proteins in corresponding fractions. Intracellular biosynthetic proteins of both gene clusters were not only detected in the cytosolic, but also in the enriched membrane fraction, indicating that the biosynthesis of acarbose and putative pyochelin metabolites takes place at the inner membrane. Actinoplanes sp. SE50/110 is a natural producer of the α-glucosidase inhibitor acarbose, a bacterial secondary metabolite that is used as a drug for the treatment of type 2 diabetes, a disease which is a global pandemic that currently affects 387 million people and accounts for 11% of worldwide healthcare expenditures (www.idf.org). The work presented here is the first comprehensive investigation of protein localization and abundance in Actinoplanes sp. SE50/110 and provides an extensive source of information for the selection of genes for future mutational analysis and other hypothesis driven experiments. The conclusion that acarbose or pyochelin family siderophores are synthesized at the inner side of the cytoplasmic membrane determined from this work, indicates that studying corresponding intermediates will be challenging. In addition to previous studies on the genome and transcriptome, the work presented here demonstrates that the next omic level, the proteome, is now accessible for detailed physiological analysis of Actinoplanes sp. SE50/110, as well as mutants derived from this and related species. Copyright © 2015 Elsevier B.V. All rights reserved.

Core Proteomic Analysis of Unique Metabolic Pathways of Salmonella enterica for the Identification of Potential Drug Targets.

PubMed

Uddin, Reaz; Sufian, Muhammad

2016-01-01

Infections caused by Salmonella enterica, a Gram-negative facultative anaerobic bacteria belonging to the family of Enterobacteriaceae, are major threats to the health of humans and animals. The recent availability of complete genome data of pathogenic strains of the S. enterica gives new avenues for the identification of drug targets and drug candidates. We have used the genomic and metabolic pathway data to identify pathways and proteins essential to the pathogen and absent from the host. We took the whole proteome sequence data of 42 strains of S. enterica and Homo sapiens along with KEGG-annotated metabolic pathway data, clustered proteins sequences using CD-HIT, identified essential genes using DEG database and discarded S. enterica homologs of human proteins in unique metabolic pathways (UMPs) and characterized hypothetical proteins with SVM-prot and InterProScan. Through this core proteomic analysis we have identified enzymes essential to the pathogen. The identification of 73 enzymes common in 42 strains of S. enterica is the real strength of the current study. We proposed all 73 unexplored enzymes as potential drug targets against the infections caused by the S. enterica. The study is comprehensive around S. enterica and simultaneously considered every possible pathogenic strain of S. enterica. This comprehensiveness turned the current study significant since, to the best of our knowledge it is the first subtractive core proteomic analysis of the unique metabolic pathways applied to any pathogen for the identification of drug targets. We applied extensive computational methods to shortlist few potential drug targets considering the druggability criteria e.g. Non-homologous to the human host, essential to the pathogen and playing significant role in essential metabolic pathways of the pathogen (i.e. S. enterica). In the current study, the subtractive proteomics through a novel approach was applied i.e. by considering only proteins of the unique metabolic pathways of the pathogens and mining the proteomic data of all completely sequenced strains of the pathogen, thus improving the quality and application of the results. We believe that the sharing of the knowledge from this study would eventually lead to bring about novel and unique therapeutic regimens against the infections caused by the S. enterica.

Functional proteomic analysis of corticosteroid pharmacodynamics in rat liver: Relationship to hepatic stress, signaling, energy regulation, and drug metabolism.

PubMed

Ayyar, Vivaswath S; Almon, Richard R; DuBois, Debra C; Sukumaran, Siddharth; Qu, Jun; Jusko, William J

2017-05-08

Corticosteroids (CS) are anti-inflammatory agents that cause extensive pharmacogenomic and proteomic changes in multiple tissues. An understanding of the proteome-wide effects of CS in liver and its relationships to altered hepatic and systemic physiology remains incomplete. Here, we report the application of a functional pharmacoproteomic approach to gain integrated insight into the complex nature of CS responses in liver in vivo. An in-depth functional analysis was performed using rich pharmacodynamic (temporal-based) proteomic data measured over 66h in rat liver following a single dose of methylprednisolone (MPL). Data mining identified 451 differentially regulated proteins. These proteins were analyzed on the basis of temporal regulation, cellular localization, and literature-mined functional information. Of the 451 proteins, 378 were clustered into six functional groups based on major clinically-relevant effects of CS in liver. MPL-responsive proteins were highly localized in the mitochondria (20%) and cytosol (24%). Interestingly, several proteins were related to hepatic stress and signaling processes, which appear to be involved in secondary signaling cascades and in protecting the liver from CS-induced oxidative damage. Consistent with known adverse metabolic effects of CS, several rate-controlling enzymes involved in amino acid metabolism, gluconeogenesis, and fatty-acid metabolism were altered by MPL. In addition, proteins involved in the metabolism of endogenous compounds, xenobiotics, and therapeutic drugs including cytochrome P450 and Phase-II enzymes were differentially regulated. Proteins related to the inflammatory acute-phase response were up-regulated in response to MPL. Functionally-similar proteins showed large diversity in their temporal profiles, indicating complex mechanisms of regulation by CS. Clinical use of corticosteroid (CS) therapy is frequent and chronic. However, current knowledge on the proteome-level effects of CS in liver and other tissues is sparse. While transcriptomic regulation following methylprednisolone (MPL) dosing has been temporally examined in rat liver, proteomic assessments are needed to better characterize the tissue-specific functional aspects of MPL actions. This study describes a functional pharmacoproteomic analysis of dynamic changes in MPL-regulated proteins in liver and provides biological insight into how steroid-induced perturbations on a molecular level may relate to both adverse and therapeutic responses presented clinically. Copyright © 2017 Elsevier B.V. All rights reserved.

Porphyrin-based Photocatalytic Nanolithography

PubMed Central

Bearinger, Jane P.; Stone, Gary; Dugan, Lawrence C.; El Dasher, Bassem; Stockton, Cheryl; Conway, James W.; Kuenzler, Tobias; Hubbell, Jeffrey A.

2009-01-01

Nanoarray fabrication is a multidisciplinary endeavor encompassing materials science, chemical engineering, and biology. We formed nanoarrays via a new technique, porphyrin-based photocatalytic nanolithography. The nanoarrays, with controlled features as small as 200 nm, exhibited regularly ordered patterns and may be appropriate for (a) rapid and parallel proteomics screening of immobilized biomolecules, (b) protein-protein interactions, and/or (c) biophysical and molecular biology studies involving spatially dictated ligand placement. We demonstrated protein immobilization utilizing nanoarrays fabricated via photocatalytic nanolithography on silicon substrates where the immobilized proteins are surrounded by a non-fouling polymer background. PMID:19406753

Preparation of monoclonal antibody bank against whole water-soluble proteins from rapid-growing bamboo shoots.

PubMed

Wu, Yu-Jen; Chen, Han-Min; Wu, Tai-Tse; Wu, Jiann-Shing; Chu, Rea-Min; Juang, Rong-Huay

2006-11-01

An antibody bank against the whole proteins in a proteome is a useful tool for biological research. Using the standard cell fusion method, and a modified screening protocol, we produced an mAb bank against the total water-soluble proteins extracted from the rapid-growing green bamboo shoots. An improved two-stage strategy was employed to enrich those poor immunogenic or lower expressed proteins. Totally, we obtained a bank of 192 mAb which were identified as distinctive to each other by 2-DE and immunostaining.

Emerging techniques for the discovery and validation of therapeutic targets for skeletal diseases.

PubMed

Cho, Christine H; Nuttall, Mark E

2002-12-01

Advances in genomics and proteomics have revolutionised the drug discovery process and target validation. Identification of novel therapeutic targets for chronic skeletal diseases is an extremely challenging process based on the difficulty of obtaining high-quality human diseased versus normal tissue samples. The quality of tissue and genomic information obtained from the sample is critical to identifying disease-related genes. Using a genomics-based approach, novel genes or genes with similar homology to existing genes can be identified from cDNA libraries generated from normal versus diseased tissue. High-quality cDNA libraries are prepared from uncontaminated homogeneous cell populations harvested from tissue sections of interest. Localised gene expression analysis and confirmation are obtained through in situ hybridisation or immunohistochemical studies. Cells overexpressing the recombinant protein are subsequently designed for primary cell-based high-throughput assays that are capable of screening large compound banks for potential hits. Afterwards, secondary functional assays are used to test promising compounds. The same overexpressing cells are used in the secondary assay to test protein activity and functionality as well as screen for small-molecule agonists or antagonists. Once a hit is generated, a structure-activity relationship of the compound is optimised for better oral bioavailability and pharmacokinetics allowing the compound to progress into development. Parallel efforts from proteomics, as well as genetics/transgenics, bioinformatics and combinatorial chemistry, and improvements in high-throughput automation technologies, allow the drug discovery process to meet the demands of the medicinal market. This review discusses and illustrates how different approaches are incorporated into the discovery and validation of novel targets and, consequently, the development of potentially therapeutic agents in the areas of osteoporosis and osteoarthritis. While current treatments exist in the form of hormone replacement therapy, antiresorptive and anabolic agents for osteoporosis, there are no disease-modifying therapies for the treatment of the most common human joint disease, osteoarthritis. A massive market potential for improved options with better safety and efficacy still remains. Therefore, the application of genomics and proteomics for both diseases should provide much needed novel therapeutic approaches to treating these major world health problems.

Distant plant homologues: don't throw out the baby.

PubMed

Gardiner, John; Overall, Robyn; Marc, Jan

2012-03-01

Plants and metazoans share many similarities in terms of conserved proteins. Antibodies have been used extensively to detect remote homologues, many of which are yet to be identified conclusively. Genome sequencing and the creation of novel sequence or structure comparison programs have assisted greatly in the identification of distant protein homologues. The continuing development of new software algorithms and the combining of bioinformatics with proteomics offer hope that remaining homologues will be soon identified. Copyright © 2011 Elsevier Ltd. All rights reserved.

Mass spectrometry and renal calculi

PubMed Central

Purcarea, VL; Sisu, I; Sisu, E

2010-01-01

The present review represents a concise and complete survey of the literature covering 2004–2009, concerning the mass spectrometric techniques involved in the structural investigation of renal calculi. After a short presentation of the fundamental mass spectrometric techniques (MALDI–TOF, QTOF, MS–MS) as well as hyphenated methods (GC–MS, LC–MS, CE–MS), an extensive study of the urinary proteome analysis as well as the detection and quantification by mass spectrometry of toxins, drugs and metabolites from renal calculi is presented. PMID:20968197

Analysis of the interactome of the Ser/Thr Protein Phosphatase type 1 in Plasmodium falciparum.

PubMed

Hollin, Thomas; De Witte, Caroline; Lenne, Astrid; Pierrot, Christine; Khalife, Jamal

2016-03-17

Protein Phosphatase 1 (PP1) is an enzyme essential to cell viability in the malaria parasite Plasmodium falciparum (Pf). The activity of PP1 is regulated by the binding of regulatory subunits, of which there are up to 200 in humans, but only 3 have been so far reported for the parasite. To better understand the P. falciparum PP1 (PfPP1) regulatory network, we here report the use of three strategies to characterize the PfPP1 interactome: co-affinity purified proteins identified by mass spectrometry, yeast two-hybrid (Y2H) screening and in silico analysis of the P. falciparum predicted proteome. Co-affinity purification followed by MS analysis identified 6 PfPP1 interacting proteins (Pips) of which 3 contained the RVxF consensus binding, 2 with a Fxx[RK]x[RK] motif, also shown to be a PP1 binding motif and one with both binding motifs. The Y2H screens identified 134 proteins of which 30 present the RVxF binding motif and 20 have the Fxx[RK]x[RK] binding motif. The in silico screen of the Pf predicted proteome using a consensus RVxF motif as template revealed the presence of 55 potential Pips. As further demonstration, 35 candidate proteins were validated as PfPP1 interacting proteins in an ELISA-based assay. To the best of our knowledge, this is the first study on PfPP1 interactome. The data reports several conserved PP1 interacting proteins as well as a high number of specific interactors to PfPP1. Their analysis indicates a high diversity of biological functions for PP1 in Plasmodium. Based on the present data and on an earlier study of the Pf interactome, a potential implication of Pips in protein folding/proteolysis, transcription and pathogenicity networks is proposed. The present work provides a starting point for further studies on the structural basis of these interactions and their functions in P. falciparum.

ExPASy: SIB bioinformatics resource portal.

PubMed

Artimo, Panu; Jonnalagedda, Manohar; Arnold, Konstantin; Baratin, Delphine; Csardi, Gabor; de Castro, Edouard; Duvaud, Séverine; Flegel, Volker; Fortier, Arnaud; Gasteiger, Elisabeth; Grosdidier, Aurélien; Hernandez, Céline; Ioannidis, Vassilios; Kuznetsov, Dmitry; Liechti, Robin; Moretti, Sébastien; Mostaguir, Khaled; Redaschi, Nicole; Rossier, Grégoire; Xenarios, Ioannis; Stockinger, Heinz

2012-07-01

ExPASy (http://www.expasy.org) has worldwide reputation as one of the main bioinformatics resources for proteomics. It has now evolved, becoming an extensible and integrative portal accessing many scientific resources, databases and software tools in different areas of life sciences. Scientists can henceforth access seamlessly a wide range of resources in many different domains, such as proteomics, genomics, phylogeny/evolution, systems biology, population genetics, transcriptomics, etc. The individual resources (databases, web-based and downloadable software tools) are hosted in a 'decentralized' way by different groups of the SIB Swiss Institute of Bioinformatics and partner institutions. Specifically, a single web portal provides a common entry point to a wide range of resources developed and operated by different SIB groups and external institutions. The portal features a search function across 'selected' resources. Additionally, the availability and usage of resources are monitored. The portal is aimed for both expert users and people who are not familiar with a specific domain in life sciences. The new web interface provides, in particular, visual guidance for newcomers to ExPASy.

Connecting G protein signaling to chemoattractant-mediated cell polarity and cytoskeletal reorganization.

PubMed

Liu, Youtao; Lacal, Jesus; Firtel, Richard A; Kortholt, Arjan

2018-07-04

The directional movement toward extracellular chemical gradients, a process called chemotaxis, is an important property of cells. Central to eukaryotic chemotaxis is the molecular mechanism by which chemoattractant-mediated activation of G-protein coupled receptors (GPCRs) induces symmetry breaking in the activated downstream signaling pathways. Studies with mainly Dictyostelium and mammalian neutrophils as experimental systems have shown that chemotaxis is mediated by a complex network of signaling pathways. Recently, several labs have used extensive and efficient proteomic approaches to further unravel this dynamic signaling network. Together these studies showed the critical role of the interplay between heterotrimeric G-protein subunits and monomeric G proteins in regulating cytoskeletal rearrangements during chemotaxis. Here we highlight how these proteomic studies have provided greater insight into the mechanisms by which the heterotrimeric G protein cycle is regulated, how heterotrimeric G proteins-induced symmetry breaking is mediated through small G protein signaling, and how symmetry breaking in G protein signaling subsequently induces cytoskeleton rearrangements and cell migration.

Application of Tandem Two-Dimensional Mass Spectrometry for Top-Down Deep Sequencing of Calmodulin

NASA Astrophysics Data System (ADS)

Floris, Federico; Chiron, Lionel; Lynch, Alice M.; Barrow, Mark P.; Delsuc, Marc-André; O'Connor, Peter B.

2018-06-01

Two-dimensional mass spectrometry (2DMS) involves simultaneous acquisition of the fragmentation patterns of all the analytes in a mixture by correlating their precursor and fragment ions by modulating precursor ions systematically through a fragmentation zone. Tandem two-dimensional mass spectrometry (MS/2DMS) unites the ultra-high accuracy of Fourier transform ion cyclotron resonance (FT-ICR) MS/MS and the simultaneous data-independent fragmentation of 2DMS to achieve extensive inter-residue fragmentation of entire proteins. 2DMS was recently developed for top-down proteomics (TDP), and applied to the analysis of calmodulin (CaM), reporting a cleavage coverage of about 23% using infrared multiphoton dissociation (IRMPD) as fragmentation technique. The goal of this work is to expand the utility of top-down protein analysis using MS/2DMS in order to extend the cleavage coverage in top-down proteomics further into the interior regions of the protein. In this case, using MS/2DMS, the cleavage coverage of CaM increased from 23% to 42%.

Targeted Proteomics Approach for Precision Plant Breeding.

PubMed

Chawade, Aakash; Alexandersson, Erik; Bengtsson, Therese; Andreasson, Erik; Levander, Fredrik

2016-02-05

Selected reaction monitoring (SRM) is a targeted mass spectrometry technique that enables precise quantitation of hundreds of peptides in a single run. This technique provides new opportunities for multiplexed protein biomarker measurements. For precision plant breeding, DNA-based markers have been used extensively, but the potential of protein biomarkers has not been exploited. In this work, we developed an SRM marker panel with assays for 104 potato (Solanum tuberosum) peptides selected using univariate and multivariate statistics. Thereafter, using random forest classification, the prediction markers were identified for Phytopthora infestans resistance in leaves, P. infestans resistance in tubers, and plant yield in potato leaf secretome samples. The results suggest that the marker panel has the predictive potential for three traits, two of which have no commercial DNA markers so far. Furthermore, the marker panel was also tested and found to be applicable to potato clones not used during the marker development. The proposed workflow is thus a proof-of-concept for targeted proteomics as an efficient readout in accelerated breeding for complex and agronomically important traits.

2-DE Compared with iTRAQ-based Proteomic Analysis of the Functional Regulation of Proteins in Rhodococcus sp. BAP-1 Response to Fluoranthene

NASA Astrophysics Data System (ADS)

Xu, Jie; Wang, Hongqi; Kong, Dekang

2018-01-01

Although the degradation pathways of Polycyclic aromatic hydrocarbons (PAHs) have been extensively studied in many bacteria, the variations in the expression levels of the key functional regulation of proteins during catabolism are still not quantitatively understood. In this study, we compared two proteomic methods, that one is two-dimensional gel electrophoresis (2-DE), a traditional widely used way and the other is isobaric tags for relative and absolute quantization (iTRAQ), an innovative approach, in order to analyze the functional regulation at the protein level in high effective fluoranthene-degrading bacteria named Rhodococcus sp. BAP-1. The number of differentially expressed proteins identified using iTRAQ is much larger than employing 2-DE. Response to fluoranthene, the key over expressed proteins in BAP-1 were NADPH-dependent FMN reductase, 30S ribosomal protein S2, S-ribosylhomocysteinase, etc.; the significant down-regulated proteins were cytochrome ubiquinol oxidase subunit, NAD(P) transhydrogenase subunit alpha, 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase, et al.

A Proteome-wide Fission Yeast Interactome Reveals Network Evolution Principles from Yeasts to Human.

PubMed

Vo, Tommy V; Das, Jishnu; Meyer, Michael J; Cordero, Nicolas A; Akturk, Nurten; Wei, Xiaomu; Fair, Benjamin J; Degatano, Andrew G; Fragoza, Robert; Liu, Lisa G; Matsuyama, Akihisa; Trickey, Michelle; Horibata, Sachi; Grimson, Andrew; Yamano, Hiroyuki; Yoshida, Minoru; Roth, Frederick P; Pleiss, Jeffrey A; Xia, Yu; Yu, Haiyuan

2016-01-14

Here, we present FissionNet, a proteome-wide binary protein interactome for S. pombe, comprising 2,278 high-quality interactions, of which ∼ 50% were previously not reported in any species. FissionNet unravels previously unreported interactions implicated in processes such as gene silencing and pre-mRNA splicing. We developed a rigorous network comparison framework that accounts for assay sensitivity and specificity, revealing extensive species-specific network rewiring between fission yeast, budding yeast, and human. Surprisingly, although genes are better conserved between the yeasts, S. pombe interactions are significantly better conserved in human than in S. cerevisiae. Our framework also reveals that different modes of gene duplication influence the extent to which paralogous proteins are functionally repurposed. Finally, cross-species interactome mapping demonstrates that coevolution of interacting proteins is remarkably prevalent, a result with important implications for studying human disease in model organisms. Overall, FissionNet is a valuable resource for understanding protein functions and their evolution. Copyright © 2016 Elsevier Inc. All rights reserved.

Transcriptomic and proteomic landscape of mitochondrial dysfunction reveals secondary coenzyme Q deficiency in mammals

PubMed Central

Atanassov, Ilian; Kuznetsova, Irina; Hinze, Yvonne; Mourier, Arnaud; Filipovska, Aleksandra

2017-01-01

Dysfunction of the oxidative phosphorylation (OXPHOS) system is a major cause of human disease and the cellular consequences are highly complex. Here, we present comparative analyses of mitochondrial proteomes, cellular transcriptomes and targeted metabolomics of five knockout mouse strains deficient in essential factors required for mitochondrial DNA gene expression, leading to OXPHOS dysfunction. Moreover, we describe sequential protein changes during post-natal development and progressive OXPHOS dysfunction in time course analyses in control mice and a middle lifespan knockout, respectively. Very unexpectedly, we identify a new response pathway to OXPHOS dysfunction in which the intra-mitochondrial synthesis of coenzyme Q (ubiquinone, Q) and Q levels are profoundly decreased, pointing towards novel possibilities for therapy. Our extensive omics analyses provide a high-quality resource of altered gene expression patterns under severe OXPHOS deficiency comparing several mouse models, that will deepen our understanding, open avenues for research and provide an important reference for diagnosis and treatment. PMID:29132502

Mammary molecular portraits reveal lineage-specific features and progenitor cell vulnerabilities.

PubMed

Casey, Alison E; Sinha, Ankit; Singhania, Rajat; Livingstone, Julie; Waterhouse, Paul; Tharmapalan, Pirashaanthy; Cruickshank, Jennifer; Shehata, Mona; Drysdale, Erik; Fang, Hui; Kim, Hyeyeon; Isserlin, Ruth; Bailey, Swneke; Medina, Tiago; Deblois, Genevieve; Shiah, Yu-Jia; Barsyte-Lovejoy, Dalia; Hofer, Stefan; Bader, Gary; Lupien, Mathieu; Arrowsmith, Cheryl; Knapp, Stefan; De Carvalho, Daniel; Berman, Hal; Boutros, Paul C; Kislinger, Thomas; Khokha, Rama

2018-06-19

The mammary epithelium depends on specific lineages and their stem and progenitor function to accommodate hormone-triggered physiological demands in the adult female. Perturbations of these lineages underpin breast cancer risk, yet our understanding of normal mammary cell composition is incomplete. Here, we build a multimodal resource for the adult gland through comprehensive profiling of primary cell epigenomes, transcriptomes, and proteomes. We define systems-level relationships between chromatin-DNA-RNA-protein states, identify lineage-specific DNA methylation of transcription factor binding sites, and pinpoint proteins underlying progesterone responsiveness. Comparative proteomics of estrogen and progesterone receptor-positive and -negative cell populations, extensive target validation, and drug testing lead to discovery of stem and progenitor cell vulnerabilities. Top epigenetic drugs exert cytostatic effects; prevent adult mammary cell expansion, clonogenicity, and mammopoiesis; and deplete stem cell frequency. Select drugs also abrogate human breast progenitor cell activity in normal and high-risk patient samples. This integrative computational and functional study provides fundamental insight into mammary lineage and stem cell biology. © 2018 Casey et al.

Detailed proteomic analysis on DM: insight into its hypoallergenicity.

PubMed

Bertino, Enrico; Gastaldi, Daniela; Monti, Giovanna; Baro, Cristina; Fortunato, Donatella; Perono Garoffo, Lorenza; Coscia, Alessandra; Fabris, Claudio; Mussap, Michele; Conti, Amedeo

2010-01-01

Successful therapy in cow milk (CM) protein allergy rests upon completely eliminating CM proteins from the child's diet: it is thus necessary to provide a replacement food. Donkey milk (DM) has recently aroused scientific and clinical interest, above all among paediatric allergologists. A deeper knowledge of proteins in DM is necessary to evaluate the immunological and physiological properties of this natural substitute for cow's milk. The paper offers a detailed comparative analysis among the protein fractions of DM, CM and human milk, following an extensive proteomic study of the casein and whey proteins of DM performed by narrow pH range 2-DE. The detailed protein composition and structural features reported in this study provide insight into the molecular reasons for the hypoallergenicity of DM. Whole DM might constitute a valid substitute of CM in feeding children with CM protein allergy and it might also constitute the basis for formulas suitable for allergic subjects in the first year of life.

Systems Proteomics View of the Endogenous Human Claudin Protein Family

PubMed Central

Liu, Fei; Koval, Michael; Ranganathan, Shoba; Fanayan, Susan; Hancock, William S.; Lundberg, Emma K.; Beavis, Ronald C.; Lane, Lydie; Duek, Paula; McQuade, Leon; Kelleher, Neil L.; Baker, Mark S.

2016-01-01

Claudins are the major transmembrane protein components of tight junctions in human endothelia and epithelia. Tissue-specific expression of claudin members suggests that this protein family is not only essential for sustaining the role of tight junctions in cell permeability control but also vital in organizing cell contact signaling by protein–protein interactions. How this protein family is collectively processed and regulated is key to understanding the role of junctional proteins in preserving cell identity and tissue integrity. The focus of this review is to first provide a brief overview of the functional context, on the basis of the extensive body of claudin biology research that has been thoroughly reviewed, for endogenous human claudin members and then ascertain existing and future proteomics techniques that may be applicable to systematically characterizing the chemical forms and interacting protein partners of this protein family in human. The ability to elucidate claudin-based signaling networks may provide new insight into cell development and differentiation programs that are crucial to tissue stability and manipulation. PMID:26680015

Plasmodium vivax trophozoite-stage proteomes

PubMed Central

Anderson, D.C.; Lapp, Stacey A.; Akinyi, Sheila; Meyer, Esmeralda V.S.; Barnwell, John W.; Korir-Morrison, Cindy; Galinski, Mary R.

2015-01-01

Plasmodium vivax is the causative infectious agent of 80–300 million annual cases of malaria. Many aspects of this parasite’s biology remain unknown. To further elucidate the interaction of P. vivax with its Saimiri boliviensis host, we obtained detailed proteomes of infected red blood cells, representing the trophozoite-enriched stage of development. Data from two of three biological replicate proteomes, emphasized here, were analyzed using five search engines, which enhanced identifications and resulted in the most comprehensive P. vivax proteomes to date, with 1375 P. vivax and 3209 S. boliviensis identified proteins. Ribosome subunit proteins were noted for both P. vivax and S. boliviensis, consistent with P. vivax’s known reticulocyte host–cell specificity. A majority of the host and pathogen proteins identified belong to specific functional categories, and several parasite gene families, while 33% of the P. vivax proteins have no reported function. Hemoglobin was significantly oxidized in both proteomes, and additional protein oxidation and nitration was detected in one of the two proteomes. Detailed analyses of these post-translational modifications are presented. The proteins identified here significantly expand the known P. vivax proteome and complexity of available host protein functionality underlying the host–parasite interactive biology, and reveal unsuspected oxidative modifications that may impact protein function. Biological significance Plasmodium vivax malaria is a serious neglected disease, causing an estimated 80 to 300 million cases annually in 95 countries. Infection can result in significant morbidity and possible death. P. vivax, unlike the much better-studied Plasmodium falciparum species, cannot be grown in long-term culture, has a dormant form in the liver called the hypnozoite stage, has a reticulocyte host–cell preference in the blood, and creates caveolae vesicle complexes at the surface of the infected reticulocyte membranes. Studies of stage-specific P. vivax expressed proteomes have been limited in scope and focused mainly on pathogen proteins, thus limiting understanding of the biology of this pathogen and its host interactions. Here three P. vivax proteomes are reported from biological replicates based on purified trophozoite-infected reticulocytes from different Saimiri boliviensis infections (the main non-human primate experimental model for P. vivax biology and pathogenesis). An in-depth analysis of two of the proteomes using 2D LC/MS/MS and multiple search engines identified 1375 pathogen proteins and 3209 host proteins. Numerous functional categories of both host and pathogen proteins were identified, including several known P. vivax protein family members (e.g., PHIST, eTRAMP and VIR), and 33% of protein identifications were classified as hypothetical. Ribosome subunit proteins were noted for both P. vivax and S. boliviensis, consistent with this parasite species’ known reticulocyte host–cell specificity. In two biological replicates analyzed for post-translational modifications, hemoglobin was extensively oxidized, and various other proteins were also oxidized or nitrated in one of the two replicates. The cause of such protein modification remains to be determined but could include oxidized heme and oxygen radicals released from the infected red blood cell’s parasite-induced acidic digestive vacuoles. In any case, the data suggests the presence of distinct infection-specific conditions whereby both the pathogen and host infected red blood cell proteins may be subject to significant oxidative stress. PMID:25545414

Regional Screening Levels (RSLs) - User's Guide (November 2017)

EPA Pesticide Factsheets

Regional Screening Level RSL user guide provides extensive guidance for risk assessors doing Chemical Risk Assessment preliminary remediation goal PRG risk based concentration RBC and rick calculations for the assess

Laser assisted microdissection, an efficient technique to understand tissue specific gene expression patterns and functional genomics in plants.

PubMed

Gautam, Vibhav; Sarkar, Ananda K

2015-04-01

Laser assisted microdissection (LAM) is an advanced technology used to perform tissue or cell-specific expression profiling of genes and proteins, owing to its ability to isolate the desired tissue or cell type from a heterogeneous population. Due to the specificity and high efficiency acquired during its pioneering use in medical science, the LAM technique has quickly been adopted for use in many biological researches. Today, it has become a potent tool to address a wide range of questions in diverse field of plant biology. Beginning with comparative transcriptome analysis of different tissues such as reproductive parts, meristems, lateral organs, roots etc., LAM has also been extensively used in plant-pathogen interaction studies, proteomics, and metabolomics. In combination with next generation sequencing and proteomics analysis, LAM has opened up promising opportunities in the area of large scale functional studies in plants. Ever since the advent of this technique, significant improvements have been achieved in term of its instrumentation and method, which has made LAM a more efficient tool applicable in wider research areas. Here, we discuss the advancement of LAM technique with special emphasis on its methodology and highlight its scope in modern research areas of plant biology. Although we put emphasis on use of LAM in transcriptome studies, which is mostly used, we also discuss its recent application and scope in proteome and metabolome studies.

Comparative proteomic and physiological analyses reveal the protective effect of exogenous polyamines in the bermudagrass (Cynodon dactylon) response to salt and drought stresses.

PubMed

Shi, Haitao; Ye, Tiantian; Chan, Zhulong

2013-11-01

Polyamines conferred enhanced abiotic stress tolerance in multiple plant species. However, the effect of polyamines on abiotic stress and physiological change in bermudagrass, the most widely used warm-season turfgrasses, are unknown. In this study, pretreatment of exogenous polyamine conferred increased salt and drought tolerances in bermudagrass. Comparative proteomic analysis was performed to further investigate polyamines mediated responses, and 36 commonly regulated proteins by at least two types of polyamines in bermudagrass were successfully identified, including 12 proteins with increased level, 20 proteins with decreased level and other 4 specifically expressed proteins. Among them, proteins involved in electron transport and energy pathways were largely enriched, and nucleoside diphosphate kinase (NDPK) and three antioxidant enzymes were extensively regulated by polyamines. Dissection of reactive oxygen species (ROS) levels indicated that polyamine-derived H2O2 production might play dual roles under abiotic stress conditions. Moreover, accumulation of osmolytes was also observed after application of exogenous polyamines, which is consistent with proteomics results that several proteins involved in carbon fixation pathway were mediated commonly by polyamines pretreatment. Taken together, we proposed that polyamines could activate multiple pathways that enhance bermudagrass adaption to salt and drought stresses. These findings might be applicable for genetically engineering of grasses and crops to improve stress tolerance.

Proteomic Analysis of Tendon Extracellular Matrix Reveals Disease Stage-specific Fragmentation and Differential Cleavage of COMP (Cartilage Oligomeric Matrix Protein)*

PubMed Central

Dakin, Stephanie Georgina; Smith, Roger Kenneth Whealands; Heinegård, Dick; Önnerfjord, Patrik; Khabut, Areej; Dudhia, Jayesh

2014-01-01

During inflammatory processes the extracellular matrix (ECM) is extensively remodeled, and many of the constituent components are released as proteolytically cleaved fragments. These degradative processes are better documented for inflammatory joint diseases than tendinopathy even though the pathogenesis has many similarities. The aims of this study were to investigate the proteomic composition of injured tendons during early and late disease stages to identify disease-specific cleavage patterns of the ECM protein cartilage oligomeric matrix protein (COMP). In addition to characterizing fragments released in naturally occurring disease, we hypothesized that stimulation of tendon explants with proinflammatory mediators in vitro would induce fragments of COMP analogous to natural disease. Therefore, normal tendon explants were stimulated with IL-1β and prostaglandin E2, and their effects on the release of COMP and its cleavage patterns were characterized. Analyses of injured tendons identified an altered proteomic composition of the ECM at all stages post injury, showing protein fragments that were specific to disease stage. IL-1β enhanced the proteolytic cleavage and release of COMP from tendon explants, whereas PGE2 had no catabolic effect. Of the cleavage fragments identified in early stage tendon disease, two fragments were generated by an IL-1-mediated mechanism. These fragments provide a platform for the development of neo-epitope assays specific to injury stage for tendon disease. PMID:24398684

Grade-Dependent Metabolic Reprogramming in Kidney Cancer Revealed by Combined Proteomics and Metabolomics Analysis [Combined Proteomics and Metabolomics Analysis Reveals Grade-Dependent Metabolism Pathways in Kidney Cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wettersten, Hiromi I.; Hakimi, A. Ari; Morin, Dexter

Kidney cancer [or renal cell carcinoma (RCC)] is known as “the internist's tumor” because it has protean systemic manifestations, suggesting that it utilizes complex, nonphysiologic metabolic pathways. Given the increasing incidence of this cancer and its lack of effective therapeutic targets, we undertook an extensive analysis of human RCC tissue employing combined grade-dependent proteomics and metabolomics analysis to determine how metabolic reprogramming occurring in this disease allows it to escape available therapeutic approaches. After validation experiments in RCC cell lines that were wild-type or mutant for the Von Hippel–Lindau tumor suppressor, in characterizing higher-grade tumors, we found that the Warburgmore » effect is relatively more prominent at the expense of the tricarboxylic acid cycle and oxidative metabolism in general. Further, we found that the glutamine metabolism pathway acts to inhibit reactive oxygen species, as evidenced by an upregulated glutathione pathway, whereas the β-oxidation pathway is inhibited, leading to increased fatty acylcarnitines. In support of findings from previous urine metabolomics analyses, we also documented tryptophan catabolism associated with immune suppression, which was highly represented in RCC compared with other metabolic pathways. Altogether, our results offer a rationale to evaluate novel antimetabolic treatment strategies being developed in other disease settings as therapeutic strategies in RCC« less

Targeted quantitative analysis of Streptococcus pyogenes virulence factors by multiple reaction monitoring.

PubMed

Lange, Vinzenz; Malmström, Johan A; Didion, John; King, Nichole L; Johansson, Björn P; Schäfer, Juliane; Rameseder, Jonathan; Wong, Chee-Hong; Deutsch, Eric W; Brusniak, Mi-Youn; Bühlmann, Peter; Björck, Lars; Domon, Bruno; Aebersold, Ruedi

2008-08-01

In many studies, particularly in the field of systems biology, it is essential that identical protein sets are precisely quantified in multiple samples such as those representing differentially perturbed cell states. The high degree of reproducibility required for such experiments has not been achieved by classical mass spectrometry-based proteomics methods. In this study we describe the implementation of a targeted quantitative approach by which predetermined protein sets are first identified and subsequently quantified at high sensitivity reliably in multiple samples. This approach consists of three steps. First, the proteome is extensively mapped out by multidimensional fractionation and tandem mass spectrometry, and the data generated are assembled in the PeptideAtlas database. Second, based on this proteome map, peptides uniquely identifying the proteins of interest, proteotypic peptides, are selected, and multiple reaction monitoring (MRM) transitions are established and validated by MS2 spectrum acquisition. This process of peptide selection, transition selection, and validation is supported by a suite of software tools, TIQAM (Targeted Identification for Quantitative Analysis by MRM), described in this study. Third, the selected target protein set is quantified in multiple samples by MRM. Applying this approach we were able to reliably quantify low abundance virulence factors from cultures of the human pathogen Streptococcus pyogenes exposed to increasing amounts of plasma. The resulting quantitative protein patterns enabled us to clearly define the subset of virulence proteins that is regulated upon plasma exposure.

Integrated pipeline for mass spectrometry-based discovery and confirmation of biomarkers demonstrated in a mouse model of breast cancer.

PubMed

Whiteaker, Jeffrey R; Zhang, Heidi; Zhao, Lei; Wang, Pei; Kelly-Spratt, Karen S; Ivey, Richard G; Piening, Brian D; Feng, Li-Chia; Kasarda, Erik; Gurley, Kay E; Eng, Jimmy K; Chodosh, Lewis A; Kemp, Christopher J; McIntosh, Martin W; Paulovich, Amanda G

2007-10-01

Despite their potential to impact diagnosis and treatment of cancer, few protein biomarkers are in clinical use. Biomarker discovery is plagued with difficulties ranging from technological (inability to globally interrogate proteomes) to biological (genetic and environmental differences among patients and their tumors). We urgently need paradigms for biomarker discovery. To minimize biological variation and facilitate testing of proteomic approaches, we employed a mouse model of breast cancer. Specifically, we performed LC-MS/MS of tumor and normal mammary tissue from a conditional HER2/Neu-driven mouse model of breast cancer, identifying 6758 peptides representing >700 proteins. We developed a novel statistical approach (SASPECT) for prioritizing proteins differentially represented in LC-MS/MS datasets and identified proteins over- or under-represented in tumors. Using a combination of antibody-based approaches and multiple reaction monitoring-mass spectrometry (MRM-MS), we confirmed the overproduction of multiple proteins at the tissue level, identified fibulin-2 as a plasma biomarker, and extensively characterized osteopontin as a plasma biomarker capable of early disease detection in the mouse. Our results show that a staged pipeline employing shotgun-based comparative proteomics for biomarker discovery and multiple reaction monitoring for confirmation of biomarker candidates is capable of finding novel tissue and plasma biomarkers in a mouse model of breast cancer. Furthermore, the approach can be extended to find biomarkers relevant to human disease.

Surface modified capillary electrophoresis combined with in solution isoelectric focusing and MALDI-TOF/TOF MS: a gel-free multidimensional electrophoresis approach for proteomic profiling--exemplified on human follicular fluid.

PubMed

Hanrieder, Jörg; Zuberovic, Aida; Bergquist, Jonas

2009-04-24

Development of miniaturized analytical tools continues to be of great interest to face the challenges in proteomic analysis of complex biological samples such as human body fluids. In the light of these challenges, special emphasis is put on the speed and simplicity of newly designed technological approaches as well as the need for cost efficiency and low sample consumption. In this study, we present an alternative multidimensional bottom-up approach for proteomic profiling for fast, efficient and sensitive protein analysis in complex biological matrices. The presented setup was based on sample pre-fractionation using microscale in solution isoelectric focusing (IEF) followed by tryptic digestion and subsequent capillary electrophoresis (CE) coupled off-line to matrix assisted laser desorption/ionization time of flight tandem mass spectrometry (MALDI TOF MS/MS). For high performance CE-separation, PolyE-323 modified capillaries were applied to minimize analyte-wall interactions. The potential of the analytical setup was demonstrated on human follicular fluid (hFF) representing a typical complex human body fluid with clinical implication. The obtained results show significant identification of 73 unique proteins (identified at 95% significance level), including mostly acute phase proteins but also protein identities that are well known to be extensively involved in follicular development.

Comparative analysis of the alveolar macrophage proteome in ALI/ARDS patients between the exudative phase and recovery phase

PubMed Central

2013-01-01

Background Despite decades of extensive studies, the morbidity and mortality for acute lung injury/acute respiratory distress syndrome (ALI/ARDS) remained high. Particularly, biomarkers essential for its early diagnosis and prognosis are lacking. Methods Recent studies suggest that alveolar macrophages (AMs) at the exudative phase of ALI/ARDS initiate, amplify and perpetuate inflammatory responses, while they resolve inflammation in the recovery phase to prevent further tissue injury and perpetuated inflammation in the lung. Therefore, proteins relevant to this functional switch could be valuable biomarkers for ALI/ARDS diagnosis and prognosis. We thus conducted comparative analysis of the AM proteome to assess its dynamic proteomic changes during ALI/ARDS progression and recovery. Results 135 proteins were characterized to be differentially expressed between AMs at the exudative and recovery phase. MALDI-TOF-MS and peptide mass fingerprint (PMF) analysis characterized 27 informative proteins, in which 17 proteins were found with a marked increase at the recovery phase, while the rest of 10 proteins were manifested by the significantly higher levels of expression at the exudative phase. Conclusions Given the role of above identified proteins played in the regulation of inflammatory responses, cell skeleton organization, oxidative stress, apoptosis and metabolism, they have the potential to serve as biomarkers for early diagnosis and prognosis in the setting of patients with ALI/ARDS. PMID:23773529

Grade-Dependent Metabolic Reprogramming in Kidney Cancer Revealed by Combined Proteomics and Metabolomics Analysis [Combined Proteomics and Metabolomics Analysis Reveals Grade-Dependent Metabolism Pathways in Kidney Cancer

DOE PAGES

Wettersten, Hiromi I.; Hakimi, A. Ari; Morin, Dexter; ...

2015-05-07

Kidney cancer [or renal cell carcinoma (RCC)] is known as “the internist's tumor” because it has protean systemic manifestations, suggesting that it utilizes complex, nonphysiologic metabolic pathways. Given the increasing incidence of this cancer and its lack of effective therapeutic targets, we undertook an extensive analysis of human RCC tissue employing combined grade-dependent proteomics and metabolomics analysis to determine how metabolic reprogramming occurring in this disease allows it to escape available therapeutic approaches. After validation experiments in RCC cell lines that were wild-type or mutant for the Von Hippel–Lindau tumor suppressor, in characterizing higher-grade tumors, we found that the Warburgmore » effect is relatively more prominent at the expense of the tricarboxylic acid cycle and oxidative metabolism in general. Further, we found that the glutamine metabolism pathway acts to inhibit reactive oxygen species, as evidenced by an upregulated glutathione pathway, whereas the β-oxidation pathway is inhibited, leading to increased fatty acylcarnitines. In support of findings from previous urine metabolomics analyses, we also documented tryptophan catabolism associated with immune suppression, which was highly represented in RCC compared with other metabolic pathways. Altogether, our results offer a rationale to evaluate novel antimetabolic treatment strategies being developed in other disease settings as therapeutic strategies in RCC« less

Proteomic Analysis of Interactions between a Deep-Sea Thermophilic Bacteriophage and Its Host at High Temperature ▿ †

PubMed Central

Wei, Dahai; Zhang, Xiaobo

2010-01-01

The virus-host interaction is essential to understanding the role that viruses play in ecological and geochemical processes in deep-sea vent ecosystems. Virus-induced changes in cellular gene expression and host physiology have been studied extensively. However, the molecular mechanism of interaction between a bacteriophage and its host at high temperature remains poorly understood. In the present study, the virus-induced gene expression profile of Geobacillus sp. E263, a thermophile isolated from a deep-sea hydrothermal ecosystem, was characterized. Based on proteomic analysis and random arbitrarily primed PCR (RAP-PCR) of Geobacillus sp. E263 cultured under non-bacteriophage GVE2 infection and GVE2 infection conditions, there were two types of protein/gene profiles in response to GVE2 infection. Twenty differentially expressed genes and proteins were revealed that could be grouped into 3 different categories based on cellular function, suggesting a coordinated response to infection. These differentially expressed genes and proteins were further confirmed by Northern blot analysis. To characterize the host proteins in response to virus infection, aspartate aminotransferase (AST) was inactivated to construct the AST mutant of Geobacillus sp. E263. The results showed that the AST protein was essential in virus infection. Thus, transcriptional and proteomic analyses and functional analysis revealed previously unknown host responses to deep-sea thermophilic virus infection. PMID:20015994

Analysis of secreted proteins from Aspergillus flavus.

PubMed

Medina, Martha L; Haynes, Paul A; Breci, Linda; Francisco, Wilson A

2005-08-01

MS/MS techniques in proteomics make possible the identification of proteins from organisms with little or no genome sequence information available. Peptide sequences are obtained from tandem mass spectra by matching peptide mass and fragmentation information to protein sequence information from related organisms, including unannotated genome sequence data. This peptide identification data can then be grouped and reconstructed into protein data. In this study, we have used this approach to study protein secretion by Aspergillus flavus, a filamentous fungus for which very little genome sequence information is available. A. flavus is capable of degrading the flavonoid rutin (quercetin 3-O-glycoside), as the only source of carbon via an extracellular enzyme system. In this continuing study, a proteomic analysis was used to identify secreted proteins from A. flavus when grown on rutin. The growth media glucose and potato dextrose were used to identify differentially expressed secreted proteins. The secreted proteins were analyzed by 1- and 2-DE and MS/MS. A total of 51 unique A. flavus secreted proteins were identified from the three growth conditions. Ten proteins were unique to rutin-, five to glucose- and one to potato dextrose-grown A. flavus. Sixteen secreted proteins were common to all three media. Fourteen identifications were of hypothetical proteins or proteins of unknown functions. To our knowledge, this is the first extensive proteomic study conducted to identify the secreted proteins from a filamentous fungus.

Screening vs. non-screening detected colorectal cancer: Differences in pre-therapeutic work up and treatment.

PubMed

Saraste, D; Martling, A; Nilsson, P J; Blom, J; Törnberg, S; Janson, M

2017-06-01

Objectives To compare preoperative staging, multidisciplinary team-assessment, and treatment in patients with screening detected and non-screening detected colorectal cancer. Methods Data on patient and tumour characteristics, staging, multidisciplinary team-assessment and treatment in patients with screening and non-screening detected colorectal cancer from 2008 to 2012 were collected from the Stockholm-Gotland screening register and the Swedish Colorectal Cancer Registry. Results The screening group had a higher proportion of stage I disease (41 vs. 15%; p < 0.001), a more complete staging of primary tumour and metastases and were more frequently multidisciplinary team-assessed than the non-screening group ( p < 0.001). In both groups, patients with endoscopically resected cancers were less completely staged and multidisciplinary team-assessed than patients with surgically resected cancers ( p < 0.001). No statistically significant differences were observed between the screening and non-screening groups in the use of neoadjuvant treatment in rectal cancer (68 vs.76%), surgical treatment with local excision techniques in stage I rectal cancer (6 vs. 9%) or adjuvant chemotherapy in stages II and III disease (46 vs. 52%). Emergency interventions for colorectal cancer occurred in 4% of screening participants vs. 11% of non-compliers. Conclusions Screening detected cancer patients were staged and multidisciplinary team assessed more extensively than patients with non-screening detected cancers. Staging and multidisciplinary team assessment prior to endoscopic resection was less complete compared with surgical resection. Extensive surgical and (neo)adjuvant treatment was given in stage I disease. Participation in screening reduced the risk of emergency surgery for colorectal cancer.

Quantitative Proteomic Analysis of Duck Ovarian Follicles Infected with Duck Tembusu Virus by Label-Free LC-MS

PubMed Central

Han, Kaikai; Zhao, Dongmin; Liu, Yuzhuo; Liu, Qingtao; Huang, Xinmei; Yang, Jing; An, Fengjiao; Li, Yin

2016-01-01

Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that has caused massive economic losses to the duck industry in China. DTMUV infection mainly results in significant decreases in egg production in egg-laying ducks within 1–2 weeks post infection. However, information on the comparative protein expression of host tissues in response to DTMUV infection is limited. In the present study, the cellular protein response to DTMUV infection in duck ovarian follicles was analyzed using nano-flow high-performance liquid chromatography-electrospray tandem mass spectrometry. Quantitative proteomic analysis revealed 131 differentially expressed proteins, among which 53 were up regulated and 78 were down regulated. The identified proteins were involved in the regulation of essential processes such as cellular structure and integrity, RNA processing, protein biosynthesis and modification, vesicle transport, signal transduction, and mitochondrial pathway. Some selected proteins that were found to be regulated in DTMUV-infected tissues were screened by quantitative real-time PCR to examine their regulation at the transcriptional level, western blot analysis was used to validate the changes of some selected proteins on translational level. To our knowledge, this study is the first to analyze the proteomic changes in duck ovarian follicles following DTMUV infection. The protein-related information obtained in this study may be useful to understand the host response to DTMUV infection and the inherent mechanism of DTMUV replication and pathogenicity. PMID:27066001

Whole CMV Proteome Pattern Recognition Analysis after HSCT Identifies Unique Epitope Targets Associated with the CMV Status

PubMed Central

Pérez-Bercoff, Lena; Valentini, Davide; Gaseitsiwe, Simani; Mahdavifar, Shahnaz; Schutkowski, Mike; Poiret, Thomas; Pérez-Bercoff, Åsa; Ljungman, Per; Maeurer, Markus J.

2014-01-01

Cytomegalovirus (CMV) infection represents a vital complication after Hematopoietic Stem Cell Transplantation (HSCT). We screened the entire CMV proteome to visualize the humoral target epitope-focus profile in serum after HSCT. IgG profiling from four patient groups (donor and/or recipient +/− for CMV) was performed at 6, 12 and 24 months after HSCT using microarray slides containing 17174 of 15mer-peptides overlapping by 4 aa covering 214 proteins from CMV. Data were analyzed using maSigPro, PAM and the ‘exclusive recognition analysis (ERA)’ to identify unique CMV epitope responses for each patient group. The ‘exclusive recognition analysis’ of serum epitope patterns segregated best 12 months after HSCT for the D+/R+ group (versus D−/R−). Epitopes were derived from UL123 (IE1), UL99 (pp28), UL32 (pp150), this changed at 24 months to 2 strongly recognized peptides provided from UL123 and UL100. Strongly (IgG) recognized CMV targets elicited also robust cytokine production in T-cells from patients after HSCT defined by intracellular cytokine staining (IL-2, TNF, IFN and IL-17). High-content peptide microarrays allow epitope profiling of entire viral proteomes; this approach can be useful to map relevant targets for diagnostics and therapy in patients with well defined clinical endpoints. Peptide microarray analysis visualizes the breadth of B-cell immune reconstitution after HSCT and provides a useful tool to gauge immune reconstitution. PMID:24740411

Proteomics of gliomas: Initial biomarker discovery and evolution of technology

PubMed Central

Kalinina, Juliya; Peng, Junmin; Ritchie, James C.; Van Meir, Erwin G.

2011-01-01

Gliomas are a group of aggressive brain tumors that diffusely infiltrate adjacent brain tissues, rendering them largely incurable, even with multiple treatment modalities and agents. Mostly asymptomatic at early stages, they present in several subtypes with astrocytic or oligodendrocytic features and invariably progress to malignant forms. Gliomas are difficult to classify precisely because of interobserver variability during histopathologic grading. Identifying biological signatures of each glioma subtype through protein biomarker profiling of tumor or tumor-proximal fluids is therefore of high priority. Such profiling not only may provide clues regarding tumor classification but may identify clinical biomarkers and pathologic targets for the development of personalized treatments. In the past decade, differential proteomic profiling techniques have utilized tumor, cerebrospinal fluid, and plasma from glioma patients to identify the first candidate diagnostic, prognostic, predictive, and therapeutic response markers, highlighting the potential for glioma biomarker discovery. The number of markers identified, however, has been limited, their reproducibility between studies is unclear, and none have been validated for clinical use. Recent technological advancements in methodologies for high-throughput profiling, which provide easy access, rapid screening, low sample consumption, and accurate protein identification, are anticipated to accelerate brain tumor biomarker discovery. Reliable tools for biomarker verification forecast translation of the biomarkers into clinical diagnostics in the foreseeable future. Herein we update the reader on the recent trends and directions in glioma proteomics, including key findings and established and emerging technologies for analysis, together with challenges we are still facing in identifying and verifying potential glioma biomarkers. PMID:21852429

PARALLEL ASSAY OF OXYGEN EQUILIBRIA OF HEMOGLOBIN

PubMed Central

Lilly, Laura E.; Blinebry, Sara K.; Viscardi, Chelsea M.; Perez, Luis; Bonaventura, Joe; McMahon, Tim J.

2013-01-01

Methods to systematically analyze in parallel the function of multiple protein or cell samples in vivo or ex vivo (i.e. functional proteomics) in a controlled gaseous environment have thus far been limited. Here we describe an apparatus and procedure that enables, for the first time, parallel assay of oxygen equilibria in multiple samples. Using this apparatus, numerous simultaneous oxygen equilibrium curves (OECs) can be obtained under truly identical conditions from blood cell samples or purified hemoglobins (Hbs). We suggest that the ability to obtain these parallel datasets under identical conditions can be of immense value, both to biomedical researchers and clinicians who wish to monitor blood health, and to physiologists studying non-human organisms and the effects of climate change on these organisms. Parallel monitoring techniques are essential in order to better understand the functions of critical cellular proteins. The procedure can be applied to human studies, wherein an OEC can be analyzed in light of an individual’s entire genome. Here, we analyzed intraerythrocytic Hb, a protein that operates at the organism’s environmental interface and then comes into close contact with virtually all of the organism’s cells. The apparatus is theoretically scalable, and establishes a functional proteomic screen that can be correlated with genomic information on the same individuals. This new method is expected to accelerate our general understanding of protein function, an increasingly challenging objective as advances in proteomic and genomic throughput outpace the ability to study proteins’ functional properties. PMID:23827235

Medicare Cancer Screening in the Context of Clinical Guidelines: 2000 to 2012.

PubMed

Maroongroge, Sean; Yu, James B

2018-04-01

Cancer screening is a ubiquitous and controversial public health issue, particularly in the elderly population. Despite extensive evidence-based guidelines for screening, it is unclear how cancer screening has changed in the Medicare population over time. We characterize trends in cancer screening for the most common cancer types in the Medicare fee-for-service (FFS) program in the context of conflicting guidelines from 2000 to 2012. We performed a descriptive analysis of retrospective claims data from the Medicare FFS program based on billing codes. Our data include all claims for Medicare part B beneficiaries who received breast, colorectal (CRC), or prostate cancer screening from 2000 to 2012 based on billing codes. We utilize a Monte Carlo permutation method to detect changes in screening trends. In total, 231,416,732 screening tests were analyzed from 2000 to 2012, representing an average of 436.8 tests per 1000 beneficiaries per year. Mammography rates declined 7.4%, with digital mammography extensively replacing film. CRC cancer screening rates declined overall. As a percentage of all CRC screening tests, colonoscopy grew from 32% to 71%. Prostate screening rates increased 16% from 2000 to 2007, and then declined to 7% less than its 2000 rate by 2012. Both the aggressiveness of screening guidelines and screening rates for the Medicare FFS population peaked and then declined from 2000 to 2012. However, guideline publications did not consistently precede utilization trend shifts. Technology adoption, practical and financial concerns, and patient preferences may have also contributed to the observed trends. Further research should be performed on the impact of multiple, conflicting guidelines in cancer screening.

Paul Pinsky, PhD | Division of Cancer Prevention

Cancer.gov

Dr. Paul Pinsky is the chief of the Early Detection Research Branch. He has a background in statistics, epidemiology and mathematical modeling. He has worked extensively with data from the Branch's two large screening trials, the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial and the National Lung Screening Trial (NLST). |

Proteomic and Biochemical Studies of Lysine Malonylation Suggest Its Malonic Aciduria-associated Regulatory Role in Mitochondrial Function and Fatty Acid Oxidation*

PubMed Central

Colak, Gozde; Pougovkina, Olga; Dai, Lunzhi; Tan, Minjia; te Brinke, Heleen; Huang, He; Cheng, Zhongyi; Park, Jeongsoon; Wan, Xuelian; Liu, Xiaojing; Yue, Wyatt W.; Wanders, Ronald J. A.; Locasale, Jason W.; Lombard, David B.; de Boer, Vincent C. J.; Zhao, Yingming

2015-01-01

The protein substrates of sirtuin 5-regulated lysine malonylation (Kmal) remain unknown, hindering its functional analysis. In this study, we carried out proteomic screening, which identified 4042 Kmal sites on 1426 proteins in mouse liver and 4943 Kmal sites on 1822 proteins in human fibroblasts. Increased malonyl-CoA levels in malonyl-CoA decarboxylase (MCD)-deficient cells induces Kmal levels in substrate proteins. We identified 461 Kmal sites showing more than a 2-fold increase in response to MCD deficiency as well as 1452 Kmal sites detected only in MCD−/− fibroblast but not MCD+/+ cells, suggesting a pathogenic role of Kmal in MCD deficiency. Cells with increased lysine malonylation displayed impaired mitochondrial function and fatty acid oxidation, suggesting that lysine malonylation plays a role in pathophysiology of malonic aciduria. Our study establishes an association between Kmal and a genetic disease and offers a rich resource for elucidating the contribution of the Kmal pathway and malonyl-CoA to cellular physiology and human diseases. PMID:26320211

Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Applications in Quantitative Proteomics.

PubMed

Chahrour, Osama; Malone, John

2017-01-01

Recent advances in inductively coupled plasma mass spectrometry (ICP-MS) hyphenated to different separation techniques have promoted it as a valuable tool in protein/peptide quantification. These emerging ICP-MS applications allow absolute quantification by measuring specific elemental responses. One approach quantifies elements already present in the structure of the target peptide (e.g. phosphorus and sulphur) as natural tags. Quantification of these natural tags allows the elucidation of the degree of protein phosphorylation in addition to absolute protein quantification. A separate approach is based on utilising bi-functional labelling substances (those containing ICP-MS detectable elements), that form a covalent chemical bond with the protein thus creating analogs which are detectable by ICP-MS. Based on the previously established stoichiometries of the labelling reagents, quantification can be achieved. This technique is very useful for the design of precise multiplexed quantitation schemes to address the challenges of biomarker screening and discovery. This review discusses the capabilities and different strategies to implement ICP-MS in the field of quantitative proteomics. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Proteomics shows new faces for the old penicillin producer Penicillium chrysogenum.

PubMed

Barreiro, Carlos; Martín, Juan F; García-Estrada, Carlos

2012-01-01

Fungi comprise a vast group of microorganisms including the Ascomycota (majority of all described fungi), the Basidiomycota (mushrooms or higher fungi), and the Zygomycota and Chytridiomycota (basal or lower fungi) that produce industrially interesting secondary metabolites, such as β-lactam antibiotics. These compounds are one of the most commonly prescribed drugs world-wide. Since Fleming's initial discovery of Penicillium notatum 80 years ago, the role of Penicillium as an antimicrobial source became patent. After the isolation of Penicillium chrysogenum NRRL 1951 six decades ago, classical mutagenesis and screening programs led to the development of industrial strains with increased productivity (at least three orders of magnitude). The new "omics" era has provided the key to understand the underlying mechanisms of the industrial strain improvement process. The review of different proteomics methods applied to P. chrysogenum has revealed that industrial modification of this microorganism was a consequence of a careful rebalancing of several metabolic pathways. In addition, the secretome analysis of P. chrysogenum has opened the door to new industrial applications for this versatile filamentous fungus.

Seminal plasma as a diagnostic fluid for male reproductive system disorders.

PubMed

Drabovich, Andrei P; Saraon, Punit; Jarvi, Keith; Diamandis, Eleftherios P

2014-05-01

Molecular biomarkers hold promise to advance the noninvasive diagnosis of male reproductive system disorders and facilitate the identification and management of these conditions through screening, early diagnosis and more accurate prognosis. Seminal plasma has great potential as a proximal fluid for protein biomarker discovery and as a clinical sample for noninvasive diagnostics. The seminal plasma proteome contains thousands of proteins and includes a large number of tissue-specific proteins that might accurately indicate a pathological process in the tissue of origin. Potential protein biomarkers for male reproductive system disorders are more abundant in seminal plasma than in blood serum or urine, and, therefore, are more easily identified and quantified in semen by mass spectrometry and other techniques. These methods have enabled elaboration of the composition of the seminal plasma proteome and the tissue specificity of seminal plasma proteins. Strategies have been developed to discover protein biomarkers in seminal plasma through integrated 'omics' approaches. Biomarkers of male infertility and prostate cancer are now emerging, and it is evident that seminal plasma has the potential to complement other diagnostic tools available in urology clinics.

A Novel Immunogenic Spore Coat-Associated Protein in Bacillus Anthracis: Characterization via Proteomics Approaches and a Vector-Based Vaccine System

PubMed Central

Liu, Yu-Tsueng; Lin, Shwu-Bin; Huang, Cheng-Po; Huang, Chun-Ming

2007-01-01

New generation anthrax vaccines have been actively explored with the aim of enhancing efficacies and decreasing undesirable side effects that could be caused by licensed vaccines. Targeting novel antigens and/or eliminating the requirements for multiple needle injections and adjuvants are major objectives in the development of new anthrax vaccines. Using proteomics approaches, we identified a spore coat-associated protein (SCAP) in Bacillus anthracis. An E. coli vector-based vaccine system was used to determine the immunogenicity of SCAP. Mice generated detectable SCAP antibodies three weeks after intranasal immunization with an intact particle of ultraviolet (UV)-irradiated E. coli vector overproducing SCAP. The production of SCAP antibodies was detected via western blotting and SCAP-spotted antigen-arrays. The adjuvant effect of a UV-irradiated E. coli vector eliminates the necessity of boosting and the use of other immunomodulators which will foster the screening and manufacturing of new generation anthrax vaccines. More importantly, the immunogenic SCAP may potentially be a new candidate for the development of anthrax vaccines. PMID:18029197

Trifunctional lipid probes for comprehensive studies of single lipid species in living cells

PubMed Central

Nadler, André; Haberkant, Per; Kirkpatrick, Joanna; Schifferer, Martina; Stein, Frank; Hauke, Sebastian; Porter, Forbes D.; Schultz, Carsten

2017-01-01

Lipid-mediated signaling events regulate many cellular processes. Investigations of the complex underlying mechanisms are difficult because several different methods need to be used under varying conditions. Here we introduce multifunctional lipid derivatives to study lipid metabolism, lipid−protein interactions, and intracellular lipid localization with a single tool per target lipid. The probes are equipped with two photoreactive groups to allow photoliberation (uncaging) and photo–cross-linking in a sequential manner, as well as a click-handle for subsequent functionalization. We demonstrate the versatility of the design for the signaling lipids sphingosine and diacylglycerol; uncaging of the probe for these two species triggered calcium signaling and intracellular protein translocation events, respectively. We performed proteomic screens to map the lipid-interacting proteome for both lipids. Finally, we visualized a sphingosine transport deficiency in patient-derived Niemann−Pick disease type C fibroblasts by fluorescence as well as correlative light and electron microscopy, pointing toward the diagnostic potential of such tools. We envision that this type of probe will become important for analyzing and ultimately understanding lipid signaling events in a comprehensive manner. PMID:28154130

Nucleic Acids for Ultra-Sensitive Protein Detection

PubMed Central

Janssen, Kris P. F.; Knez, Karel; Spasic, Dragana; Lammertyn, Jeroen

2013-01-01

Major advancements in molecular biology and clinical diagnostics cannot be brought about strictly through the use of genomics based methods. Improved methods for protein detection and proteomic screening are an absolute necessity to complement to wealth of information offered by novel, high-throughput sequencing technologies. Only then will it be possible to advance insights into clinical processes and to characterize the importance of specific protein biomarkers for disease detection or the realization of “personalized medicine”. Currently however, large-scale proteomic information is still not as easily obtained as its genomic counterpart, mainly because traditional antibody-based technologies struggle to meet the stringent sensitivity and throughput requirements that are required whereas mass-spectrometry based methods might be burdened by significant costs involved. However, recent years have seen the development of new biodetection strategies linking nucleic acids with existing antibody technology or replacing antibodies with oligonucleotide recognition elements altogether. These advancements have unlocked many new strategies to lower detection limits and dramatically increase throughput of protein detection assays. In this review, an overview of these new strategies will be given. PMID:23337338

Proteomics Shows New Faces for the Old Penicillin Producer Penicillium chrysogenum

PubMed Central

Barreiro, Carlos; Martín, Juan F.; García-Estrada, Carlos

2012-01-01

Fungi comprise a vast group of microorganisms including the Ascomycota (majority of all described fungi), the Basidiomycota (mushrooms or higher fungi), and the Zygomycota and Chytridiomycota (basal or lower fungi) that produce industrially interesting secondary metabolites, such as β-lactam antibiotics. These compounds are one of the most commonly prescribed drugs world-wide. Since Fleming's initial discovery of Penicillium notatum 80 years ago, the role of Penicillium as an antimicrobial source became patent. After the isolation of Penicillium chrysogenum NRRL 1951 six decades ago, classical mutagenesis and screening programs led to the development of industrial strains with increased productivity (at least three orders of magnitude). The new “omics” era has provided the key to understand the underlying mechanisms of the industrial strain improvement process. The review of different proteomics methods applied to P. chrysogenum has revealed that industrial modification of this microorganism was a consequence of a careful rebalancing of several metabolic pathways. In addition, the secretome analysis of P. chrysogenum has opened the door to new industrial applications for this versatile filamentous fungus. PMID:22318718

Proteomic Analysis of Whole Human Saliva Detects Enhanced Expression of Interleukin-1 Receptor Antagonist, Thioredoxin and Lipocalin-1 in Cigarette Smokers Compared to Non-Smokers

PubMed Central

Jessie, Kala; Pang, Wei Wei; Haji, Zubaidah; Rahim, Abdul; Hashim, Onn Haji

2010-01-01

A gel-based proteomics approach was used to screen for proteins of differential abundance between the saliva of smokers and those who had never smoked. Subjecting precipitated proteins from whole human saliva of healthy non-smokers to two-dimensional electrophoresis (2-DE) generated typical profiles comprising more than 50 proteins. While 35 of the proteins were previously established by other researchers, an additional 22 proteins were detected in the 2-DE saliva protein profiles generated in the present study. When the 2-DE profiles were compared to those obtained from subjects considered to be heavy cigarette smokers, three saliva proteins, including interleukin-1 receptor antagonist, thioredoxin and lipocalin-1, showed significant enhanced expression. The distribution patterns of lipocalin-1 isoforms were also different between cigarette smokers and non-smokers. The three saliva proteins have good potential to be used as biomarkers for the adverse effects of smoking and the risk for inflammatory and chronic diseases that are associated with it. PMID:21151451

A novel assay to assess the effect of pharmaceutical compounds on the differentiation of podocytes.

PubMed

Kindt, Frances; Hammer, Elke; Kemnitz, Stefan; Blumenthal, Antje; Klemm, Paul; Schlüter, Rabea; Quaggin, Susan E; van den Brandt, Jens; Fuellen, Georg; Völker, Uwe; Endlich, Karlhans; Endlich, Nicole

2017-01-01

Therapeutic options for treating glomerulopathies, the main cause of chronic kidney disease, are limited. Podocyte dedifferentiation is a major event in the pathogenesis of glomerulopathies. The goal of the present study was, therefore, to develop an assay to monitor podocyte differentiation suitable for compound screening. We isolated and cultured glomeruli from transgenic mice, expressing cyan fluorescent protein (CFP) under the control of the promoter of nephrin, a marker of podocyte differentiation. Mean CFP fluorescence intensity per glomerulus (MFG) was determined by summation of all glomerular voxels from confocal z-stacks in the absence and presence of pharmaceutical compounds. In untreated cultured glomeruli, MFG remained fairly stable during the first 5 days, when foot processes were already effaced, and the level of many podocyte-specific proteins was only mildly affected, as revealed by proteomics. Between day 6 and 9, MFG decreased to almost zero. The decrease in MFG was paralleled by a decrease in CFP and nephrin expression, as determined by RT-PCR, western blots and proteomics. Puromycin aminonucleoside (PAN), which damages podocytes, concentration-dependently induced a complete loss of MFG. Dexamethasone (25 μM) and pioglitazone (10 μM) markedly attenuated the effect of 0.6 μg·mL -1 PAN on MFG. In summary, we established a novel assay to assess the effect of pharmaceutical compounds on the differentiation of podocytes in situ. Our assay is suitable for compound screening to identify drugs for the treatment of glomerulopathies. © 2016 The British Pharmacological Society.

A novel assay to assess the effect of pharmaceutical compounds on the differentiation of podocytes

PubMed Central

Kindt, Frances; Hammer, Elke; Kemnitz, Stefan; Blumenthal, Antje; Klemm, Paul; Schlüter, Rabea; Quaggin, Susan E; van den Brandt, Jens; Fuellen, Georg; Völker, Uwe; Endlich, Karlhans

2016-01-01

Background and Purpose Therapeutic options for treating glomerulopathies, the main cause of chronic kidney disease, are limited. Podocyte dedifferentiation is a major event in the pathogenesis of glomerulopathies. The goal of the present study was, therefore, to develop an assay to monitor podocyte differentiation suitable for compound screening. Experimental Approach We isolated and cultured glomeruli from transgenic mice, expressing cyan fluorescent protein (CFP) under the control of the promoter of nephrin, a marker of podocyte differentiation. Mean CFP fluorescence intensity per glomerulus (MFG) was determined by summation of all glomerular voxels from confocal z‐stacks in the absence and presence of pharmaceutical compounds. Key Results In untreated cultured glomeruli, MFG remained fairly stable during the first 5 days, when foot processes were already effaced, and the level of many podocyte‐specific proteins was only mildly affected, as revealed by proteomics. Between day 6 and 9, MFG decreased to almost zero. The decrease in MFG was paralleled by a decrease in CFP and nephrin expression, as determined by RT‐PCR, western blots and proteomics. Puromycin aminonucleoside (PAN), which damages podocytes, concentration‐dependently induced a complete loss of MFG. Dexamethasone (25 μM) and pioglitazone (10 μM) markedly attenuated the effect of 0.6 μg·mL−1 PAN on MFG. Conclusion and Implications In summary, we established a novel assay to assess the effect of pharmaceutical compounds on the differentiation of podocytes in situ. Our assay is suitable for compound screening to identify drugs for the treatment of glomerulopathies. PMID:27858997

Seasonal proteomic changes reveal molecular adaptations to preserve and replenish liver proteins during ground squirrel hibernation

PubMed Central

Epperson, L. Elaine; Rose, James C.; Carey, Hannah V.

2010-01-01

Hibernators are unique among mammals in their ability to survive extended periods of time with core body temperatures near freezing and with dramatically reduced heart, respiratory, and metabolic rates in a state known as torpor. To gain insight into the molecular events underlying this remarkable physiological phenotype, we applied a proteomic screening approach to identify liver proteins that differ between the summer active (SA) and the entrance (Ent) phase of winter hibernation in 13-lined ground squirrels. The relative abundance of 1,600 protein spots separated on two-dimensional gels was quantitatively determined using fluorescence difference gel electrophoresis, and 74 unique proteins exhibiting significant differences between the two states were identified using liquid chromatography followed by tandem mass spectrometry (LC-MS/MS). Proteins elevated in Ent hibernators included liver fatty acid-binding protein, fatty acid transporter, and 3-hydroxy-3-methylglutaryl-CoA synthase, which support the known metabolic fuel switch to lipid and ketone body utilization in winter. Several proteins involved in protein stability and protein folding were also elevated in the Ent phase, consistent with previous findings. In contrast to transcript screening results, there was a surprising increase in the abundance of proteins involved in protein synthesis during Ent hibernation, including several initiation and elongation factors. This finding, coupled with decreased abundance of numerous proteins involved in amino acid and nitrogen metabolism, supports the intriguing hypothesis that the mechanism of protein preservation and resynthesis is used by hibernating ground squirrels to help avoid nitrogen toxicity and ensure preservation of essential amino acids throughout the long winter fast. PMID:19923364

Proteomics research in India: an update.

PubMed

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.

Serum Metabonomics of Mild Acute Pancreatitis.

PubMed

Xu, Hongmin; Zhang, Lei; Kang, Huan; Zhang, Jiandong; Liu, Jie; Liu, Shuye

2016-11-01

Mild acute pancreatitis (MAP) is a common acute abdominal disease, and exhibits rising incidence in recent decades. As an important component of systemic biology, metabonomics is a new discipline developed following genomics and proteomics. In this study, the objective was to analyze the serum metabonomics of patients with MAP, aiming to screen metabolic markers with potential diagnostic values. An analysis platform with ultra performance liquid chromatography-high-resolution mass spectrometry was used to screen the difference metabolites related to MAP diagnosis and disease course monitoring. A total of 432 endogenous metabolites were screened out from 122 serum samples, and 49 difference metabolites were verified, among which 12 difference metabolites were identified by nonparametric test. After material identification, eight metabolites exhibited reliable results, and their levels in MAP serum were higher than those in healthy serum. Four metabolites exhibited gradual downward trend with treatment process going on, and the differences were statistically significant (P < 0.05). Metabonomic analysis has revealed eight metabolites with potential diagnostic values toward MAP, among which four metabolites can be used to monitor the disease course. © 2016 Wiley Periodicals, Inc.

Screening the receptorome to discover the molecular targets for plant-derived psychoactive compounds: a novel approach for CNS drug discovery.

PubMed

Roth, Bryan L; Lopez, Estela; Beischel, Scott; Westkaemper, Richard B; Evans, Jon M

2004-05-01

Because psychoactive plants exert profound effects on human perception, emotion, and cognition, discovering the molecular mechanisms responsible for psychoactive plant actions will likely yield insights into the molecular underpinnings of human consciousness. Additionally, it is likely that elucidation of the molecular targets responsible for psychoactive drug actions will yield validated targets for CNS drug discovery. This review article focuses on an unbiased, discovery-based approach aimed at uncovering the molecular targets responsible for psychoactive drug actions wherein the main active ingredients of psychoactive plants are screened at the "receptorome" (that portion of the proteome encoding receptors). An overview of the receptorome is given and various in silico, public-domain resources are described. Newly developed tools for the in silico mining of data derived from the National Institute of Mental Health Psychoactive Drug Screening Program's (NIMH-PDSP) K(i) Database (K(i) DB) are described in detail. Additionally, three case studies aimed at discovering the molecular targets responsible for Hypericum perforatum, Salvia divinorum, and Ephedra sinica actions are presented. Finally, recommendations are made for future studies.

High-resolution Antibody Array Analysis of Childhood Acute Leukemia Cells*

PubMed Central

Kanderova, Veronika; Kuzilkova, Daniela; Stuchly, Jan; Vaskova, Martina; Brdicka, Tomas; Fiser, Karel; Hrusak, Ondrej; Lund-Johansen, Fridtjof

2016-01-01

Acute leukemia is a disease pathologically manifested at both genomic and proteomic levels. Molecular genetic technologies are currently widely used in clinical research. In contrast, sensitive and high-throughput proteomic techniques for performing protein analyses in patient samples are still lacking. Here, we used a technology based on size exclusion chromatography followed by immunoprecipitation of target proteins with an antibody bead array (Size Exclusion Chromatography-Microsphere-based Affinity Proteomics, SEC-MAP) to detect hundreds of proteins from a single sample. In addition, we developed semi-automatic bioinformatics tools to adapt this technology for high-content proteomic screening of pediatric acute leukemia patients. To confirm the utility of SEC-MAP in leukemia immunophenotyping, we tested 31 leukemia diagnostic markers in parallel by SEC-MAP and flow cytometry. We identified 28 antibodies suitable for both techniques. Eighteen of them provided excellent quantitative correlation between SEC-MAP and flow cytometry (p < 0.05). Next, SEC-MAP was applied to examine 57 diagnostic samples from patients with acute leukemia. In this assay, we used 632 different antibodies and detected 501 targets. Of those, 47 targets were differentially expressed between at least two of the three acute leukemia subgroups. The CD markers correlated with immunophenotypic categories as expected. From non-CD markers, we found DBN1, PAX5, or PTK2 overexpressed in B-cell precursor acute lymphoblastic leukemias, LAT, SH2D1A, or STAT5A overexpressed in T-cell acute lymphoblastic leukemias, and HCK, GLUD1, or SYK overexpressed in acute myeloid leukemias. In addition, OPAL1 overexpression corresponded to ETV6-RUNX1 chromosomal translocation. In summary, we demonstrated that SEC-MAP technology is a powerful tool for detecting hundreds of proteins in clinical samples obtained from pediatric acute leukemia patients. It provides information about protein size and reveals differences in protein expression between particular leukemia subgroups. Forty-seven of SEC-MAP identified targets were validated by other conventional method in this study. PMID:26785729

Differential Proteomic Analysis of Noncardia Gastric Cancer from Individuals of Northern Brazil

PubMed Central

Leal, Mariana Ferreira; Chung, Janete; Calcagno, Danielle Queiroz; Assumpção, Paulo Pimentel; Demachki, Samia; da Silva, Ismael Dale Cotrim Guerreiro; Chammas, Roger; Burbano, Rommel Rodríguez; de Arruda Cardoso Smith, Marília

2012-01-01

Gastric cancer is the second leading cause of cancer-related death worldwide. The identification of new cancer biomarkers is necessary to reduce the mortality rates through the development of new screening assays and early diagnosis, as well as new target therapies. In this study, we performed a proteomic analysis of noncardia gastric neoplasias of individuals from Northern Brazil. The proteins were analyzed by two-dimensional electrophoresis and mass spectrometry. For the identification of differentially expressed proteins, we used statistical tests with bootstrapping resampling to control the type I error in the multiple comparison analyses. We identified 111 proteins involved in gastric carcinogenesis. The computational analysis revealed several proteins involved in the energy production processes and reinforced the Warburg effect in gastric cancer. ENO1 and HSPB1 expression were further evaluated. ENO1 was selected due to its role in aerobic glycolysis that may contribute to the Warburg effect. Although we observed two up-regulated spots of ENO1 in the proteomic analysis, the mean expression of ENO1 was reduced in gastric tumors by western blot. However, mean ENO1 expression seems to increase in more invasive tumors. This lack of correlation between proteomic and western blot analyses may be due to the presence of other ENO1 spots that present a slightly reduced expression, but with a high impact in the mean protein expression. In neoplasias, HSPB1 is induced by cellular stress to protect cells against apoptosis. In the present study, HSPB1 presented an elevated protein and mRNA expression in a subset of gastric cancer samples. However, no association was observed between HSPB1 expression and clinicopathological characteristics. Here, we identified several possible biomarkers of gastric cancer in individuals from Northern Brazil. These biomarkers may be useful for the assessment of prognosis and stratification for therapy if validated in larger clinical study sets. PMID:22860099

Proteomics of Durum Wheat Grain during Transition to Conservation Agriculture

PubMed Central

Galieni, Angelica; Stagnari, Fabio; Bonas, Urbana; Speca, Stefano; Faccini, Andrea; Pisante, Michele; Marmiroli, Nelson

2016-01-01

Nitrogen management in combination with sustainable agronomic techniques can have a great impact on the wheat grain proteome influencing its technological quality. In this study, proteomic analyses were used to document changes in the proportion of prolamins in mature grains of the newly released Italian durum wheat cv Achille. Such an approach was applied to wheat fertilized with urea (UREA) and calcium nitrate (NITRATE), during the transition to no-till Conservation Agriculture (CA) practice in a Mediterranean environment. Results obtained in a two-years field experiment study suggest low molecular weight glutenins (LMW-GS) as the fraction particularly inducible regardless of the N-form. Quantitative analyses of LMW-GS by 2D-GE followed by protein identification by LC-ESI-MS/MS showed that the stable increase was principally due to C-type LMW-GS. The highest accumulation resulted from a physiologically healthier state of plants treated with UREA and NITRATE. Proteomic analysis on the total protein fraction during the active phase of grain filling was also performed. For both N treatments, but at different extent, an up-regulation of different classes of proteins was observed: i) enzymes involved in glycolysis and citric acid cycles which contribute to an enhanced source of energy and carbohydrates, ii) stress proteins like heat shock proteins (HSPs) and antioxidant enzymes, such as peroxidases and superoxide dismutase which protect the grain from abiotic stress during starch and storage protein synthesis. In conclusion N inputs, which combined rate with N form gave high yield and improved quality traits in the selected durum wheat cultivar. The specific up-regulation of some HSPs, antioxidant enzymes and defense proteins in the early stages of grain development and physiological indicators related to fitness traits, could be useful bio-indicators, for wheat genotype screening under more sustainable agronomic conditions, like transition phase to no-till CA in Mediterranean environments. PMID:27281174

Exploration of Spinal Cord Aging-Related Proteins Using a Proteomics Approach.

PubMed

Kamiya, Koshiro; Furuya, Takeo; Hashimoto, Masayuki; Mannoji, Chikato; Inada, Taigo; Ota, Mitsutoshi; Maki, Satoshi; Ijima, Yasushi; Saito, Junya; Kitamura, Mitsuhiro; Ohtori, Seiji; Orita, Sumihisa; Inage, Kazuhide; Yamazaki, Masashi; Koda, Masao

2017-01-01

How aging affects the spinal cord at a molecular level is unclear. The aim of this study was to explore spinal cord aging-related proteins that may be involved in pathological mechanisms of age-related changes in the spinal cord. Spinal cords of 2-year-old and 8-week-old female Sprague-Dawley rats were dissected from the animals. Protein samples were subjected to 2-dimentional polyacrylamide gel electrophoresis followed by mass spectrometry. Screened proteins were further investigated with immunohistochemistry and Western blotting. Among the screened proteins, we selected α-crystallin B-subunit (αB-crystallin) and peripherin for further investigation because these proteins were previously reported to be related to central nervous system pathologies. Immunohistochemistry and Western blotting revealed significant upregulation of αB-crystallin and peripherin expression in aged rat spinal cord. Further exploration is needed to elucidate the precise mechanism and potential role of these upregulated proteins in spinal cord aging processes.

Combinatorial Libraries of Arrayable Single-Chain Antibodies

NASA Astrophysics Data System (ADS)

Benhar, Itai

Antibodies that bind their respective targets with high affinity and specificity have proven to be essential reagents for biological research. Antibody phage display has become the leading tool for the rapid isolation of single-chain variable fragment (scFv) antibodies in vitro for research applications, but there is usually a gap between scFv isolation and its application in an array format suitable for high-throughput proteomics. In this chapter, we present our antibody phage display system where antibody isolation and scFv immobilization are facilitated by the design of the phagemid vector used as platform. In our system, the scFvs are fused at their C-termini to a cellulose-binding domain (CBD) and can be immobilized onto cellulose-based filters. This made it possible to develop a unique filter lift screen that allowed the efficient screen for multiple binding specificities, and to directly apply library-derived scFvs in an antibody spotted microarray.

Novel droplet platforms for the detection of disease biomarkers.

PubMed

Zec, Helena; Shin, Dong Jin; Wang, Tza-Huei

2014-09-01

Personalized medicine - healthcare based on individual genetic variation - has the potential to transform the way healthcare is delivered to patients. The promise of personalized medicine has been predicated on the predictive and diagnostic power of genomic and proteomic biomarkers. Biomarker screening may help improve health outcomes, for example, by identifying individuals' susceptibility to diseases and predicting how patients will respond to drugs. Microfluidic droplet technology offers an exciting opportunity to revolutionize the accessibility of personalized medicine. A framework for the role of droplet microfluidics in biomarker detection can be based on two main themes. Emulsion-based microdroplet platforms can provide new ways to measure and detect biomolecules. In addition, microdroplet platforms facilitate high-throughput screening of biomarkers. Meanwhile, surface-based droplet platforms provide an opportunity to develop miniaturized diagnostic systems. These platforms may function as portable benchtop environments that dramatically shorten the transition of a benchtop assay into a point-of-care format.

Integrating cell biology and proteomic approaches in plants.

PubMed

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 representative studies combining proteomic and cell biology methods for various purposes. Integrating cell biology approaches with proteomic ones allow validation and better interpretation of proteomic data. Moreover, cell biology methods remarkably extend the knowledge provided by proteomic studies and might be fundamental for the functional complementation of proteomic data. This review article summarizes current literature linking proteomics with cell biology. Copyright © 2017 Elsevier B.V. All rights reserved.

Compression performance of HEVC and its format range and screen content coding extensions

NASA Astrophysics Data System (ADS)

Li, Bin; Xu, Jizheng; Sullivan, Gary J.

2015-09-01

This paper presents a comparison-based test of the objective compression performance of the High Efficiency Video Coding (HEVC) standard, its format range extensions (RExt), and its draft screen content coding extensions (SCC). The current dominant standard, H.264/MPEG-4 AVC, is used as an anchor reference in the comparison. The conditions used for the comparison tests were designed to reflect relevant application scenarios and to enable a fair comparison to the maximum extent feasible - i.e., using comparable quantization settings, reference frame buffering, intra refresh periods, rate-distortion optimization decision processing, etc. It is noted that such PSNR-based objective comparisons generally provide more conservative estimates of HEVC benefit than are found in subjective studies. The experimental results show that, when compared with H.264/MPEG-4 AVC, HEVC version 1 provides a bit rate savings for equal PSNR of about 23% for all-intra coding, 34% for random access coding, and 38% for low-delay coding. This is consistent with prior studies and the general characterization that HEVC can provide about a bit rate savings of about 50% for equal subjective quality for most applications. The HEVC format range extensions provide a similar bit rate savings of about 13-25% for all-intra coding, 28-33% for random access coding, and 32-38% for low-delay coding at different bit rate ranges. For lossy coding of screen content, the HEVC screen content coding extensions achieve a bit rate savings of about 66%, 63%, and 61% for all-intra coding, random access coding, and low-delay coding, respectively. For lossless coding, the corresponding bit rate savings are about 40%, 33%, and 32%, respectively.

Development of Droplet Microfluidics Enabling High-Throughput Single-Cell Analysis.

PubMed

Wen, Na; Zhao, Zhan; Fan, Beiyuan; Chen, Deyong; Men, Dong; Wang, Junbo; Chen, Jian

2016-07-05

This article reviews recent developments in droplet microfluidics enabling high-throughput single-cell analysis. Five key aspects in this field are included in this review: (1) prototype demonstration of single-cell encapsulation in microfluidic droplets; (2) technical improvements of single-cell encapsulation in microfluidic droplets; (3) microfluidic droplets enabling single-cell proteomic analysis; (4) microfluidic droplets enabling single-cell genomic analysis; and (5) integrated microfluidic droplet systems enabling single-cell screening. We examine the advantages and limitations of each technique and discuss future research opportunities by focusing on key performances of throughput, multifunctionality, and absolute quantification.

Pharmacological screening technologies for venom peptide discovery.

PubMed

Prashanth, Jutty Rajan; Hasaballah, Nojod; Vetter, Irina

2017-12-01

Venomous animals occupy one of the most successful evolutionary niches and occur on nearly every continent. They deliver venoms via biting and stinging apparatuses with the aim to rapidly incapacitate prey and deter predators. This has led to the evolution of venom components that act at a number of biological targets - including ion channels, G-protein coupled receptors, transporters and enzymes - with exquisite selectivity and potency, making venom-derived components attractive pharmacological tool compounds and drug leads. In recent years, plate-based pharmacological screening approaches have been introduced to accelerate venom-derived drug discovery. A range of assays are amenable to this purpose, including high-throughput electrophysiology, fluorescence-based functional and binding assays. However, despite these technological advances, the traditional activity-guided fractionation approach is time-consuming and resource-intensive. The combination of screening techniques suitable for miniaturization with sequence-based discovery approaches - supported by advanced proteomics, mass spectrometry, chromatography as well as synthesis and expression techniques - promises to further improve venom peptide discovery. Here, we discuss practical aspects of establishing a pipeline for venom peptide drug discovery with a particular emphasis on pharmacology and pharmacological screening approaches. This article is part of the Special Issue entitled 'Venom-derived Peptides as Pharmacological Tools.' Copyright © 2017 Elsevier Ltd. All rights reserved.

Completed | Office of Cancer Clinical Proteomics Research

Cancer.gov

Prior to the current Clinical Proteomic Tumor Analysis Consortium (CPTAC), previously funded initiatives associated with clinical proteomics research included: Clinical Proteomic Tumor Analysis Consortium (CPTAC 2.0) Clinical Proteomic Technologies for Cancer Initiative (CPTC) Mouse Proteomic Technologies Initiative

Divergent synthesis and identification of the cellular targets of deoxyelephantopins

NASA Astrophysics Data System (ADS)

Lagoutte, Roman; Serba, Christelle; Abegg, Daniel; Hoch, Dominic G.; Adibekian, Alexander; Winssinger, Nicolas

2016-08-01

Herbal extracts containing sesquiterpene lactones have been extensively used in traditional medicine and are known to be rich in α,β-unsaturated functionalities that can covalently engage target proteins. Here we report synthetic methodologies to access analogues of deoxyelephantopin, a sesquiterpene lactone with anticancer properties. Using alkyne-tagged cellular probes and quantitative proteomics analysis, we identified several cellular targets of deoxyelephantopin. We further demonstrate that deoxyelephantopin antagonizes PPARγ activity in situ via covalent engagement of a cysteine residue in the zinc-finger motif of this nuclear receptor.

On a bender—BARs, ESCRTs, COPs, and finally getting your coat

PubMed Central

Sali, Andrej; Rout, Michael P.

2011-01-01

Tremendous variety in form and function is displayed among the intracellular membrane systems of different eukaryotes. Until recently, few clues existed as to how these internal membrane systems had originated and diversified. However, proteomic, structural, and comparative genomics studies together have revealed extensive similarities among many of the protein complexes used in controlling the morphology and trafficking of intracellular membranes. These new insights have had a profound impact on our understanding of the evolutionary origins of the internal architecture of the eukaryotic cell. PMID:21670211

Educational websites--Bioinformatics Tools II.

PubMed

Lomberk, Gwen

2009-01-01

In this issue, the highlighted websites are a continuation of a series of educational websites; this one in particular from a couple of years ago, Bioinformatics Tools [Pancreatology 2005;5:314-315]. These include sites that are valuable resources for many research needs in genomics and proteomics. Bioinformatics has become a laboratory tool to map sequences to databases, develop models of molecular interactions, evaluate structural compatibilities, describe differences between normal and disease-associated DNA, identify conserved motifs within proteins, and chart extensive signaling networks, all in silico. Copyright 2008 S. Karger AG, Basel and IAP.

Semi-quantitative proteomics of mammalian cells upon short-term exposure to non-ionizing electromagnetic fields.

PubMed

Kuzniar, Arnold; Laffeber, Charlie; Eppink, Berina; Bezstarosti, Karel; Dekkers, Dick; Woelders, Henri; Zwamborn, A Peter M; Demmers, Jeroen; Lebbink, Joyce H G; Kanaar, Roland

2017-01-01

The potential effects of non-ionizing electromagnetic fields (EMFs), such as those emitted by power-lines (in extremely low frequency range), mobile cellular systems and wireless networking devices (in radio frequency range) on human health have been intensively researched and debated. However, how exposure to these EMFs may lead to biological changes underlying possible health effects is still unclear. To reveal EMF-induced molecular changes, unbiased experiments (without a priori focusing on specific biological processes) with sensitive readouts are required. We present the first proteome-wide semi-quantitative mass spectrometry analysis of human fibroblasts, osteosarcomas and mouse embryonic stem cells exposed to three types of non-ionizing EMFs (ELF 50 Hz, UMTS 2.1 GHz and WiFi 5.8 GHz). We performed controlled in vitro EMF exposures of metabolically labeled mammalian cells followed by reliable statistical analyses of differential protein- and pathway-level regulations using an array of established bioinformatics methods. Our results indicate that less than 1% of the quantitated human or mouse proteome responds to the EMFs by small changes in protein abundance. Further network-based analysis of the differentially regulated proteins did not detect significantly perturbed cellular processes or pathways in human and mouse cells in response to ELF, UMTS or WiFi exposure. In conclusion, our extensive bioinformatics analyses of semi-quantitative mass spectrometry data do not support the notion that the short-time exposures to non-ionizing EMFs have a consistent biologically significant bearing on mammalian cells in culture.

Quantitative nucleolar proteomics reveals nuclear re-organization during stress- induced senescence in mouse fibroblast

PubMed Central

2011-01-01

Background Nucleolus is the most prominent mammalian organelle within the nucleus which is also the site for ribosomal biogenesis. There have been many reports indicating the involvement of nucleolus in the process of aging. Several proteins related to aging have been shown to localize in the nucleolus, which suggests the role of this organelle in senescence. Results In this study, we used quantitative mass spectrometry to map the flux of proteins into and out of the nucleolus during the induction of senescence in cultured mammalian cells. Changes in the abundance of 344 nucleolar proteins in sodium butyrate-induced senescence in NIH3T3 cells were studied by SILAC (stable isotope labeling by amino acids in cell culture)-based mass spectrometry. Biochemically, we have validated the proteomic results and confirmed that B23 (nucleophosmin) protein was down-regulated, while poly (ADP-ribose) polymerase (PARP) and nuclear DNA helicase II (NDH II/DHX9/RHA) were up-regulated in the nucleolus upon treatment with sodium butyrate. Accumulation of chromatin in the nucleolus was also observed, by both proteomics and microscopy, in sodium butyrate-treated cells. Similar observations were found in other models of senescence, namely, in mitoxantrone- (MTX) treated cells and primary fibroblasts from the Lamin A knockout mice. Conclusion Our data indicate an extensive nuclear organization during senescence and suggest that the redistribution of B23 protein and chromatin can be used as an important marker for senescence. PMID:21835027

N-terminal Proteomics Assisted Profiling of the Unexplored Translation Initiation Landscape in Arabidopsis thaliana *

PubMed Central

Ndah, Elvis; Jonckheere, Veronique

2017-01-01

Proteogenomics is an emerging research field yet lacking a uniform method of analysis. Proteogenomic studies in which N-terminal proteomics and ribosome profiling are combined, suggest that a high number of protein start sites are currently missing in genome annotations. We constructed a proteogenomic pipeline specific for the analysis of N-terminal proteomics data, with the aim of discovering novel translational start sites outside annotated protein coding regions. In summary, unidentified MS/MS spectra were matched to a specific N-terminal peptide library encompassing protein N termini encoded in the Arabidopsis thaliana genome. After a stringent false discovery rate filtering, 117 protein N termini compliant with N-terminal methionine excision specificity and indicative of translation initiation were found. These include N-terminal protein extensions and translation from transposable elements and pseudogenes. Gene prediction provided supporting protein-coding models for approximately half of the protein N termini. Besides the prediction of functional domains (partially) contained within the newly predicted ORFs, further supporting evidence of translation was found in the recently released Araport11 genome re-annotation of Arabidopsis and computational translations of sequences stored in public repositories. Most interestingly, complementary evidence by ribosome profiling was found for 23 protein N termini. Finally, by analyzing protein N-terminal peptides, an in silico analysis demonstrates the applicability of our N-terminal proteogenomics strategy in revealing protein-coding potential in species with well- and poorly-annotated genomes. PMID:28432195

N-terminal Proteomics Assisted Profiling of the Unexplored Translation Initiation Landscape in Arabidopsis thaliana.

PubMed

Willems, Patrick; Ndah, Elvis; Jonckheere, Veronique; Stael, Simon; Sticker, Adriaan; Martens, Lennart; Van Breusegem, Frank; Gevaert, Kris; Van Damme, Petra

2017-06-01

Proteogenomics is an emerging research field yet lacking a uniform method of analysis. Proteogenomic studies in which N-terminal proteomics and ribosome profiling are combined, suggest that a high number of protein start sites are currently missing in genome annotations. We constructed a proteogenomic pipeline specific for the analysis of N-terminal proteomics data, with the aim of discovering novel translational start sites outside annotated protein coding regions. In summary, unidentified MS/MS spectra were matched to a specific N-terminal peptide library encompassing protein N termini encoded in the Arabidopsis thaliana genome. After a stringent false discovery rate filtering, 117 protein N termini compliant with N-terminal methionine excision specificity and indicative of translation initiation were found. These include N-terminal protein extensions and translation from transposable elements and pseudogenes. Gene prediction provided supporting protein-coding models for approximately half of the protein N termini. Besides the prediction of functional domains (partially) contained within the newly predicted ORFs, further supporting evidence of translation was found in the recently released Araport11 genome re-annotation of Arabidopsis and computational translations of sequences stored in public repositories. Most interestingly, complementary evidence by ribosome profiling was found for 23 protein N termini. Finally, by analyzing protein N-terminal peptides, an in silico analysis demonstrates the applicability of our N-terminal proteogenomics strategy in revealing protein-coding potential in species with well- and poorly-annotated genomes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Proteomic and transcriptomic analysis of Arabidopsis seeds: molecular evidence for successive processing of seed proteins and its implication in the stress response to sulfur nutrition.

PubMed

Higashi, Yasuhiro; Hirai, Masami Yokota; Fujiwara, Toru; Naito, Satoshi; Noji, Masaaki; Saito, Kazuki

2006-11-01

Seed storage proteins are synthesized as sources of carbon, nitrogen and sulfur for the next generation of plants. Their composition changes according to nutritional conditions. Here, we report the precise molecular identification of seed proteins by proteomic analysis of wild-type Arabidopsis thaliana and methionine-over-accumulating mutant mto1-1 plants. The identities of 50 protein spots were determined in the protein extract of mature Arabidopsis seeds by two-dimensional (2D) gel electrophoresis and subsequent mass spectrometric analysis. Of these protein spots, 42 were identified as derived from 12S globulins or 2S albumins. These results indicate that approximately 84% of protein species in Arabidopsis seeds are derived from a few genes coding for 12S globulins and 2S albumins. Extensive mass spectrometric analysis of the 42 spots revealed that successive C-terminal degradation occurred on the 12S globulins. The feasibility of this C-terminal processing was rationalized by molecular modeling of the three-dimensional structure of 12S globulins. The C-terminal degradation at glutamic acid residues of the 12S globulin subunits was repressed under sulfur-deficient conditions. Transcriptome analysis was combined with proteomic analysis to elucidate the mechanism of changes in seed protein composition in response to sulfur deficiency. The results suggest that seed storage proteins in Arabidopsis undergo multi-layer regulation, with emphasis on post-translational modifications that enable the plant to respond to sulfur deficiency.

Performance Investigation of Proteomic Identification by HCD/CID Fragmentations in Combination with High/Low-Resolution Detectors on a Tribrid, High-Field Orbitrap Instrument

PubMed Central

Shen, Shichen; Sheng, Quanhu; Shyr, Yu; Qu, Jun

2016-01-01

The recently-introduced Orbitrap Fusion mass spectrometry permits various types of MS2 acquisition methods. To date, these different MS2 strategies and the optimal data interpretation approach for each have not been adequately evaluated. This study comprehensively investigated the four MS2 strategies: HCD-OT (higher-energy-collisional-dissociation with Orbitrap detection), HCD-IT (HCD with ion trap, IT), CID-IT (collision-induced-dissociation with IT) and CID-OT on Orbitrap Fusion. To achieve extensive comparison and identify the optimal data interpretation method for each technique, several search engines (SEQUEST and Mascot) and post-processing methods (score-based, PeptideProphet, and Percolator) were assessed for all techniques for the analysis of a human cell proteome. It was found that divergent conclusions could be made from the same dataset when different data interpretation approaches were used and therefore requiring a relatively fair comparison among techniques. Percolator was chosen for comparison of techniques because it performs the best among all search engines and MS2 strategies. For the analysis of human cell proteome using individual MS2 strategies, the highest number of identifications was achieved by HCD-OT, followed by HCD-IT and CID-IT. Based on these results, we concluded that a relatively fair platform for data interpretation is necessary to avoid divergent conclusions from the same dataset, and HCD-OT and HCD-IT may be preferable for protein/peptide identification using Orbitrap Fusion. PMID:27472422

The proteome and phosphoproteome of Neurospora crassa in response to cellulose, sucrose and carbon starvation

DOE PAGES

Xiong, Yi; Coradetti, Samuel T.; Li, Xin; ...

2014-05-29

Improving cellulolytic enzyme production by plant biomass degrading fungi holds great potential in reducing costs associated with production of next-generation biofuels generated from lignocellulose. How fungi sense cellulosic materials and respond by secreting enzymes has mainly been examined by assessing function of transcriptional regulators and via transcriptional profiling. Here, we obtained global proteomic and phosphoproteomic profiles of the plant biomass degrading filamentous fungus Neurospora crassa grown on different carbon sources, i.e. sucrose, no carbon, and cellulose, by performing isobaric tags for relative and absolute quantification (iTRAQ) -based LC-MS/MS analyses. A comparison between proteomes and transcriptomes under identical carbon conditions suggestsmore » that extensive post-transcriptional regulation occurs in N. crassa in response to exposure to cellulosic material. Several hundred amino acid residues with differential phosphorylation levels on crystalline cellulose (Avicel) or carbon-free medium versus sucrose medium were identified, including phosphorylation sites in a major transcriptional activator for cellulase genes, CLR1, as well as a cellobionic acid transporter, CBT1. Finally, we found mutation of phosphorylation sites on CLR1 did not have a major effect on transactivation of cellulase production, while mutation of phosphorylation sites in CBT1 increased its transporting capacity. Our data provides rich information at both the protein and phosphorylation levels of the early cellular responses to carbon starvation and cellulosic induction and aids in a greater understanding of the underlying post-transcriptional regulatory mechanisms in filamentous fungi.« less

Proteomics of buccal squamous cell carcinoma: the involvement of multiple pathways in tumorigenesis.

PubMed

Chen, Jia; He, Qing-Yu; Yuen, Anthony Po-Wing; Chiu, Jeng-Fu

2004-08-01

Squamous cell carcinoma (SCC) of the buccal mucosa is an aggressive oral cancer. It mainly occurs in Central and Southeast Asia, and is closely related to the practice of tobacco smoking and betel squid chewing. The high recurrence and low survival rates of buccal SCC require our continued efforts to understand the pathogenesis of the disease for designing better therapeutic strategies. We used proteomic technology to analyze buccal SCC tissues aiming at identifying tumor-associated proteins for the utilization as biomarkers or molecular targets. With the exception of alpha B-crystallin being substantially reduced, a number of proteins were found to be significantly over-expressed in cancer tissues. These increased proteins included glycolytic enzymes, heat-shock proteins, tumor antigens, cytoskeleton proteins, enzymes involved in detoxification and anti-oxidation systems, and proteins involved in mitochondrial and intracellular signaling pathways. These extensive protein variations indicate that multiple cellular pathways were involved in the process of tumorigenesis, and suggest that multiple protein molecules should be simultaneously targeted as an effective strategy to counter the disease. At least, SCC antigen, G protein, glutathione S-transferase, manganese superoxide dismutase, annexins, voltage-dependent anion channel, cyclophilin A, stratifin and galectin 7 are candidates for targeted proteins. The present findings also demonstrated that rich protein information can be produced by means of proteomic analysis for a better understanding of the oncogenesis and pathogenesis in a global way, which in turn is a basis for the rational designs of diagnostic and therapeutic methods.

Embryonic transcriptome and proteome analyses on hepatic lipid metabolism in chickens divergently selected for abdominal fat content.

PubMed

Na, Wei; Wu, Yuan-Yuan; Gong, Peng-Fei; Wu, Chun-Yan; Cheng, Bo-Han; Wang, Yu-Xiang; Wang, Ning; Du, Zhi-Qiang; Li, Hui

2018-05-23

In avian species, liver is the main site of de novo lipogenesis, and hepatic lipid metabolism relates closely to adipose fat deposition. Using our fat and lean chicken lines of striking differences in abdominal fat content, post-hatch lipid metabolism in both liver and adipose tissues has been studied extensively. However, whether molecular discrepancy for hepatic lipid metabolism exists in chicken embryos remains obscure. We performed transcriptome and proteome profiling on chicken livers at five embryonic stages (E7, E12, E14, E17 and E21) between the fat and lean chicken lines. At each stage, 521, 141, 882, 979 and 169 differentially expressed genes were found by the digital gene expression, respectively, which were significantly enriched in the metabolic, PPAR signaling and fatty acid metabolism pathways. Quantitative proteomics analysis found 20 differentially expressed proteins related to lipid metabolism, PPAR signaling, fat digestion and absorption, and oxidative phosphorylation pathways. Combined analysis showed that genes and proteins related to lipid transport (intestinal fatty acid-binding protein, nucleoside diphosphate kinase, and apolipoprotein A-I), lipid clearance (heat shock protein beta-1) and energy metabolism (NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 10 and succinate dehydrogenase flavoprotein subunit) were significantly differentially expressed between the two lines. For hepatic lipid metabolism at embryonic stages, molecular differences related to lipid transport, lipid clearance and energy metabolism exist between the fat and lean chicken lines, which might contribute to the striking differences of abdominal fat deposition at post-hatch stages.

Semi-quantitative proteomics of mammalian cells upon short-term exposure to non-ionizing electromagnetic fields

PubMed Central

Laffeber, Charlie; Eppink, Berina; Bezstarosti, Karel; Dekkers, Dick; Woelders, Henri; Zwamborn, A. Peter M.; Demmers, Jeroen; Lebbink, Joyce H. G.; Kanaar, Roland

2017-01-01

The potential effects of non-ionizing electromagnetic fields (EMFs), such as those emitted by power-lines (in extremely low frequency range), mobile cellular systems and wireless networking devices (in radio frequency range) on human health have been intensively researched and debated. However, how exposure to these EMFs may lead to biological changes underlying possible health effects is still unclear. To reveal EMF-induced molecular changes, unbiased experiments (without a priori focusing on specific biological processes) with sensitive readouts are required. We present the first proteome-wide semi-quantitative mass spectrometry analysis of human fibroblasts, osteosarcomas and mouse embryonic stem cells exposed to three types of non-ionizing EMFs (ELF 50 Hz, UMTS 2.1 GHz and WiFi 5.8 GHz). We performed controlled in vitro EMF exposures of metabolically labeled mammalian cells followed by reliable statistical analyses of differential protein- and pathway-level regulations using an array of established bioinformatics methods. Our results indicate that less than 1% of the quantitated human or mouse proteome responds to the EMFs by small changes in protein abundance. Further network-based analysis of the differentially regulated proteins did not detect significantly perturbed cellular processes or pathways in human and mouse cells in response to ELF, UMTS or WiFi exposure. In conclusion, our extensive bioinformatics analyses of semi-quantitative mass spectrometry data do not support the notion that the short-time exposures to non-ionizing EMFs have a consistent biologically significant bearing on mammalian cells in culture. PMID:28234898

Structural and metabolic transitions of C4 leaf development and differentiation defined by microscopy and quantitative proteomics in maize.

PubMed

Majeran, Wojciech; Friso, Giulia; Ponnala, Lalit; Connolly, Brian; Huang, Mingshu; Reidel, Edwin; Zhang, Cankui; Asakura, Yukari; Bhuiyan, Nazmul H; Sun, Qi; Turgeon, Robert; van Wijk, Klaas J

2010-11-01

C(4) grasses, such as maize (Zea mays), have high photosynthetic efficiency through combined biochemical and structural adaptations. C(4) photosynthesis is established along the developmental axis of the leaf blade, leading from an undifferentiated leaf base just above the ligule into highly specialized mesophyll cells (MCs) and bundle sheath cells (BSCs) at the tip. To resolve the kinetics of maize leaf development and C(4) differentiation and to obtain a systems-level understanding of maize leaf formation, the accumulation profiles of proteomes of the leaf and the isolated BSCs with their vascular bundle along the developmental gradient were determined using large-scale mass spectrometry. This was complemented by extensive qualitative and quantitative microscopy analysis of structural features (e.g., Kranz anatomy, plasmodesmata, cell wall, and organelles). More than 4300 proteins were identified and functionally annotated. Developmental protein accumulation profiles and hierarchical cluster analysis then determined the kinetics of organelle biogenesis, formation of cellular structures, metabolism, and coexpression patterns. Two main expression clusters were observed, each divided in subclusters, suggesting that a limited number of developmental regulatory networks organize concerted protein accumulation along the leaf gradient. The coexpression with BSC and MC markers provided strong candidates for further analysis of C(4) specialization, in particular transporters and biogenesis factors. Based on the integrated information, we describe five developmental transitions that provide a conceptual and practical template for further analysis. An online protein expression viewer is provided through the Plant Proteome Database.

Reductive evolution of architectural repertoires in proteomes and the birth of the tripartite world

PubMed Central

Wang, Minglei; Yafremava, Liudmila S.; Caetano-Anollés, Derek; Mittenthal, Jay E.; Caetano-Anollés, Gustavo

2007-01-01

The repertoire of protein architectures in proteomes is evolutionarily conserved and capable of preserving an accurate record of genomic history. Here we use a census of protein architecture in 185 genomes that have been fully sequenced to generate genome-based phylogenies that describe the evolution of the protein world at fold (F) and fold superfamily (FSF) levels. The patterns of representation of F and FSF architectures over evolutionary history suggest three epochs in the evolution of the protein world: (1) architectural diversification, where members of an architecturally rich ancestral community diversified their protein repertoire; (2) superkingdom specification, where superkingdoms Archaea, Bacteria, and Eukarya were specified; and (3) organismal diversification, where F and FSF specific to relatively small sets of organisms appeared as the result of diversification of organismal lineages. Functional annotation of FSF along these architectural chronologies revealed patterns of discovery of biological function. Most importantly, the analysis identified an early and extensive differential loss of architectures occurring primarily in Archaea that segregates the archaeal lineage from the ancient community of organisms and establishes the first organismal divide. Reconstruction of phylogenomic trees of proteomes reflects the timeline of architectural diversification in the emerging lineages. Thus, Archaea undertook a minimalist strategy using only a small subset of the full architectural repertoire and then crystallized into a diversified superkingdom late in evolution. Our analysis also suggests a communal ancestor to all life that was molecularly complex and adopted genomic strategies currently present in Eukarya. PMID:17908824

Large-Scale SRM Screen of Urothelial Bladder Cancer Candidate Biomarkers in Urine.

PubMed

Duriez, Elodie; Masselon, Christophe D; Mesmin, Cédric; Court, Magali; Demeure, Kevin; Allory, Yves; Malats, Núria; Matondo, Mariette; Radvanyi, François; Garin, Jérôme; Domon, Bruno

2017-04-07

Urothelial bladder cancer is a condition associated with high recurrence and substantial morbidity and mortality. Noninvasive urinary tests that would detect bladder cancer and tumor recurrence are required to significantly improve patient care. Over the past decade, numerous bladder cancer candidate biomarkers have been identified in the context of extensive proteomics or transcriptomics studies. To translate these findings in clinically useful biomarkers, the systematic evaluation of these candidates remains the bottleneck. Such evaluation involves large-scale quantitative LC-SRM (liquid chromatography-selected reaction monitoring) measurements, targeting hundreds of signature peptides by monitoring thousands of transitions in a single analysis. The design of highly multiplexed SRM analyses is driven by several factors: throughput, robustness, selectivity and sensitivity. Because of the complexity of the samples to be analyzed, some measurements (transitions) can be interfered by coeluting isobaric species resulting in biased or inconsistent estimated peptide/protein levels. Thus the assessment of the quality of SRM data is critical to allow flagging these inconsistent data. We describe an efficient and robust method to process large SRM data sets, including the processing of the raw data, the detection of low-quality measurements, the normalization of the signals for each protein, and the estimation of protein levels. Using this methodology, a variety of proteins previously associated with bladder cancer have been assessed through the analysis of urine samples from a large cohort of cancer patients and corresponding controls in an effort to establish a priority list of most promising candidates to guide subsequent clinical validation studies.

Multifocal demyelinating motor neuropathy and hamartoma syndrome associated with a de novo PTEN mutation

PubMed Central

Bansagi, Boglarka; Phan, Vietxuan; Baker, Mark R.; O'Sullivan, Julia; Jennings, Matthew J.; Whittaker, Roger G.; Müller, Juliane S.; Duff, Jennifer; Griffin, Helen; Miller, James A.L.; Gorman, Grainne S.; Lochmüller, Hanns; Chinnery, Patrick F.; Roos, Andreas; Swan, Laura E.

2018-01-01

Objective To describe a patient with a multifocal demyelinating motor neuropathy with onset in childhood and a mutation in phosphatase and tensin homolog (PTEN), a tumor suppressor gene associated with inherited tumor susceptibility conditions, macrocephaly, autism, ataxia, tremor, and epilepsy. Functional implications of this protein have been investigated in Parkinson and Alzheimer diseases. Methods We performed whole-exome sequencing in the patient's genomic DNA validated by Sanger sequencing. Immunoblotting, in vitro enzymatic assay, and label-free shotgun proteomic profiling were performed in the patient's fibroblasts. Results The predominant clinical presentation of the patient was a childhood onset, asymmetric progressive multifocal motor neuropathy. In addition, he presented with macrocephaly, autism spectrum disorder, and skin hamartomas, considered as clinical criteria for PTEN-related hamartoma tumor syndrome. Extensive tumor screening did not detect any malignancies. We detected a novel de novo heterozygous c.269T>C, p.(Phe90Ser) PTEN variant, which was absent in both parents. The pathogenicity of the variant is supported by altered expression of several PTEN-associated proteins involved in tumorigenesis. Moreover, fibroblasts showed a defect in catalytic activity of PTEN against the secondary substrate, phosphatidylinositol 3,4-trisphosphate. In support of our findings, focal hypermyelination leading to peripheral neuropathy has been reported in PTEN-deficient mice. Conclusion We describe a novel phenotype, PTEN-associated multifocal demyelinating motor neuropathy with a skin hamartoma syndrome. A similar mechanism may potentially underlie other forms of Charcot-Marie-Tooth disease with involvement of the phosphatidylinositol pathway. PMID:29720545

Efforts Aimed To Reduce Attrition in Antimalarial Drug Discovery: A Systematic Evaluation of the Current Antimalarial Targets Portfolio.

PubMed

Chaparro, María Jesús; Calderón, Félix; Castañeda, Pablo; Fernández-Alvaro, Elena; Gabarró, Raquel; Gamo, Francisco Javier; Gómez-Lorenzo, María G; Martín, Julio; Fernández, Esther

2018-04-13

Malaria remains a major global health problem. In 2015 alone, more than 200 million cases of malaria were reported, and more than 400,000 deaths occurred. Since 2010, emerging resistance to current front-line ACTs (artemisinin combination therapies) has been detected in endemic countries. Therefore, there is an urgency for new therapies based on novel modes of action, able to relieve symptoms as fast as the artemisinins and/or block malaria transmission. During the past few years, the antimalarial community has focused their efforts on phenotypic screening as a pragmatic approach to identify new hits. Optimization efforts on several chemical series have been successful, and clinical candidates have been identified. In addition, recent advances in genetics and proteomics have led to the target deconvolution of phenotypic clinical candidates. New mechanisms of action will also be critical to overcome resistance and reduce attrition. Therefore, a complementary strategy focused on identifying well-validated targets to start hit identification programs is essential to reinforce the clinical pipeline. Leveraging published data, we have assessed the status quo of the current antimalarial target portfolio with a focus on the blood stage clinical disease. From an extensive list of reported Plasmodium targets, we have defined triage criteria. These criteria consider genetic, pharmacological, and chemical validation, as well as tractability/doability, and safety implications. These criteria have provided a quantitative score that has led us to prioritize those targets with the highest probability to deliver successful and differentiated new drugs.

MALDI-TOF mass spectrometry analysis of small molecular weight compounds (under 10 KDa) as biomarkers of rat hearts undergoing arecoline challenge.

PubMed

Chen, Tung-Sheng; Chang, Mu-Hsin; Kuo, Wei-Wen; Lin, Yueh-Min; Yeh, Yu-Lan; Day, Cecilia Hsuan; Lin, Chien-Chung; Tsai, Fuu-Jen; Tsai, Chang-Hai; Huang, Chih-Yang

2013-04-01

Statistical and clinical reports indicate that betel nut chewing is strongly associated with progression of oral cancer because some ingredients in betel nuts are potential cancer promoters, especially arecoline. Early diagnosis for cancer biomarkers is the best strategy for prevention of cancer progression. Several methods are suggested for investigating cancer biomarkers. Among these methods, gel-based proteomics approach is the most powerful and recommended tool for investigating biomarkers due to its high-throughput. However, this proteomics approach is not suitable for screening biomarkers with molecular weight under 10 KDa because of the characteristics of gel electrophoresis. This study investigated biomarkers with molecular weight under 10 KDa in rats with arecoline challenge. The centrifuging vials with membrane (10 KDa molecular weight cut-off) played a crucial role in this study. After centrifuging, the filtrate (containing compounds with molecular weight under 10 KDa) was collected and spotted on a sample plate for MALDI-TOF mass spectrometry analysis. Compared to control, three extra peaks (m/z values were 1553.1611, 1668.2097 and 1740.1832, respectively) were found in sera and two extra peaks were found in heart tissue samples (408.9719 and 524.9961, respectively). These small compounds should play important roles and may be potential biomarker candidates in rats with arecoline. This study successfully reports a mass-based method for investigating biomarker candidates with small molecular weight in different types of sample (including serum and tissue). In addition, this reported method is more time-efficient (1 working day) than gel-based proteomics approach (5~7 working days).

Proteomic study of periovarian adipose tissue in 17β-estradiol-treated and untreated ovariectomized rats.

PubMed

Amengual-Cladera, Emilia; Capllonch-Amer, Gabriela; Lladó, Isabel; Gianotti, Magdalena; Proenza, Ana M

2016-04-01

Taking into account the sexual dimorphism previously found in white adipose tissue (WAT) regarding mitochondrial function and biogenesis, as well as insulin sensitivity, the aim of this study was to go further into the role of sex hormones in this dimorphism. To achieve this objective, we used ovariectomized rats and performed a screening by means of proteomic analyses of the periovarian WAT, combined with a study of the protein levels of specific factors involved in mitochondrial function. Rats were ovariectomized at 5 weeks of age and subcutaneously injected every 48 h with corn-oil (OVX group) or with 17β-estradiol (E2, 10 μg/kg body mass; OVX + E2 group) for 4 weeks prior to sacrifice. Beside proteomic analysis, protein levels of Transcription Factor A, Mitochondrial (TFAM), cytochrome oxidase (COX)II, and COXIV were determined by Western blot, and mRNA levels of peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, ERα, ERβ, lipoprotein lipase (LPL), peroxisome proliferator-activated receptor-γ (PPARγ), and adiponectin were quantified by real-time PCR. Our results show that ovariectomy leads to an increase in anabolic processes and inflammatory protein levels as well as to a decrease in some of the markers of mitochondrial function, which are restored, at least in part, by E2 supplementation. Indeed, this E2 supplementation seems to be counteracted by a decline in ERα and in the ERα to ERβ ratio values that could be directed to avoid an over-stimulation of the E2 signaling pathway, given the possibility of an activation of extra-gonadal steroid biosynthetic pathways.

Combination of Metabolomic and Proteomic Analysis Revealed Different Features among Lactobacillus delbrueckii Subspecies bulgaricus and lactis Strains While In Vivo Testing in the Model Organism Caenorhabditis elegans Highlighted Probiotic Properties

PubMed Central

Zanni, Elena; Schifano, Emily; Motta, Sara; Sciubba, Fabio; Palleschi, Claudio; Mauri, Pierluigi; Perozzi, Giuditta; Uccelletti, Daniela; Devirgiliis, Chiara; Miccheli, Alfredo

2017-01-01

Lactobacillus delbrueckii represents a technologically relevant member of lactic acid bacteria, since the two subspecies bulgaricus and lactis are widely associated with fermented dairy products. In the present work, we report the characterization of two commercial strains belonging to L. delbrueckii subspecies bulgaricus, lactis and a novel strain previously isolated from a traditional fermented fresh cheese. A phenomic approach was performed by combining metabolomic and proteomic analysis of the three strains, which were subsequently supplemented as food source to the model organism Caenorhabditis elegans, with the final aim to evaluate their possible probiotic effects. Restriction analysis of 16S ribosomal DNA revealed that the novel foodborne strain belonged to L. delbrueckii subspecies lactis. Proteomic and metabolomic approaches showed differences in folate, aminoacid and sugar metabolic pathways among the three strains. Moreover, evaluation of C. elegans lifespan, larval development, brood size, and bacterial colonization capacity demonstrated that L. delbrueckii subsp. bulgaricus diet exerted beneficial effects on nematodes. On the other hand, both L. delbrueckii subsp. lactis strains affected lifespan and larval development. We have characterized three strains belonging to L. delbrueckii subspecies bulgaricus and lactis highlighting their divergent origin. In particular, the two closely related isolates L. delbrueckii subspecies lactis display different galactose metabolic capabilities. Moreover, the L. delbrueckii subspecies bulgaricus strain demonstrated potential probiotic features. Combination of omic platforms coupled with in vivo screening in the simple model organism C. elegans is a powerful tool to characterize industrially relevant bacterial isolates. PMID:28702021

Combination of Metabolomic and Proteomic Analysis Revealed Different Features among Lactobacillus delbrueckii Subspecies bulgaricus and lactis Strains While In Vivo Testing in the Model Organism Caenorhabditis elegans Highlighted Probiotic Properties.

PubMed

Zanni, Elena; Schifano, Emily; Motta, Sara; Sciubba, Fabio; Palleschi, Claudio; Mauri, Pierluigi; Perozzi, Giuditta; Uccelletti, Daniela; Devirgiliis, Chiara; Miccheli, Alfredo

2017-01-01

Lactobacillus delbrueckii represents a technologically relevant member of lactic acid bacteria, since the two subspecies bulgaricus and lactis are widely associated with fermented dairy products. In the present work, we report the characterization of two commercial strains belonging to L. delbrueckii subspecies bulgaricus , lactis and a novel strain previously isolated from a traditional fermented fresh cheese. A phenomic approach was performed by combining metabolomic and proteomic analysis of the three strains, which were subsequently supplemented as food source to the model organism Caenorhabditis elegans , with the final aim to evaluate their possible probiotic effects. Restriction analysis of 16S ribosomal DNA revealed that the novel foodborne strain belonged to L. delbrueckii subspecies lactis . Proteomic and metabolomic approaches showed differences in folate, aminoacid and sugar metabolic pathways among the three strains. Moreover, evaluation of C. elegans lifespan, larval development, brood size, and bacterial colonization capacity demonstrated that L. delbrueckii subsp. bulgaricus diet exerted beneficial effects on nematodes. On the other hand, both L. delbrueckii subsp. lactis strains affected lifespan and larval development. We have characterized three strains belonging to L. delbrueckii subspecies bulgaricus and lactis highlighting their divergent origin. In particular, the two closely related isolates L. delbrueckii subspecies lactis display different galactose metabolic capabilities. Moreover, the L. delbrueckii subspecies bulgaricus strain demonstrated potential probiotic features. Combination of omic platforms coupled with in vivo screening in the simple model organism C. elegans is a powerful tool to characterize industrially relevant bacterial isolates.

Proteomic analysis of trans-hemispheric motor cortex reorganization following contralateral C7 nerve transfer

PubMed Central

Yuan, Yin; Xu, Xiu-yue; Lao, Jie; Zhao, Xin

2018-01-01

Nerve transfer is the most common treatment for total brachial plexus avulsion injury. After nerve transfer, the movement of the injured limb may be activated by certain movements of the healthy limb at the early stage of recovery, i.e., trans-hemispheric reorganization. Previous studies have focused on functional magnetic resonance imaging and changes in brain-derived neurotrophic factor and growth associated protein 43, but there have been no proteomics studies. In this study, we designed a rat model of total brachial plexus avulsion injury involving contralateral C7 nerve transfer. Isobaric tags for relative and absolute quantitation and western blot assay were then used to screen differentially expressed proteins in bilateral motor cortices. We found that most differentially expressed proteins in both cortices of upper limb were associated with nervous system development and function (including neuron differentiation and development, axonogenesis, and guidance), microtubule and cytoskeleton organization, synapse plasticity, and transmission of nerve impulses. Two key differentially expressed proteins, neurofilament light (NFL) and Thy-1, were identified. In contralateral cortex, the NFL level was upregulated 2 weeks after transfer and downregulated at 1 and 5 months. The Thy-1 level was upregulated from 1 to 5 months. In the affected cortex, the NFL level increased gradually from 1 to 5 months. Western blot results of key differentially expressed proteins were consistent with the proteomic findings. These results indicate that NFL and Thy-1 play an important role in trans-hemispheric organization following total brachial plexus root avulsion and contralateral C7 nerve transfer. PMID:29557385

Proteomics to study DNA-bound and chromatin-associated gene regulatory complexes

PubMed Central

Wierer, Michael; Mann, Matthias

2016-01-01

High-resolution mass spectrometry (MS)-based proteomics is a powerful method for the identification of soluble protein complexes and large-scale affinity purification screens can decode entire protein interaction networks. In contrast, protein complexes residing on chromatin have been much more challenging, because they are difficult to purify and often of very low abundance. However, this is changing due to recent methodological and technological advances in proteomics. Proteins interacting with chromatin marks can directly be identified by pulldowns with synthesized histone tails containing posttranslational modifications (PTMs). Similarly, pulldowns with DNA baits harbouring single nucleotide polymorphisms or DNA modifications reveal the impact of those DNA alterations on the recruitment of transcription factors. Accurate quantitation – either isotope-based or label free – unambiguously pinpoints proteins that are significantly enriched over control pulldowns. In addition, protocols that combine classical chromatin immunoprecipitation (ChIP) methods with mass spectrometry (ChIP-MS) target gene regulatory complexes in their in-vivo context. Similar to classical ChIP, cells are crosslinked with formaldehyde and chromatin sheared by sonication or nuclease digested. ChIP-MS baits can be proteins in tagged or endogenous form, histone PTMs, or lncRNAs. Locus-specific ChIP-MS methods would allow direct purification of a single genomic locus and the proteins associated with it. There, loci can be targeted either by artificial DNA-binding sites and corresponding binding proteins or via proteins with sequence specificity such as TAL or nuclease deficient Cas9 in combination with a specific guide RNA. We predict that advances in MS technology will soon make such approaches generally applicable tools in epigenetics. PMID:27402878

The test skeletal matrix of the black sea urchin Arbacia lixula.

PubMed

Kanold, Julia M; Immel, Francoise; Broussard, Cédric; Guichard, Nathalie; Plasseraud, Laurent; Corneillat, Marion; Alcaraz, Gérard; Brümmer, Franz; Marin, Frédéric

2015-03-01

In the field of biomineralization, the past decade has been marked by the increasing use of high throughput techniques, i.e. proteomics, for identifying in one shot the protein content of complex macromolecular mixtures extracted from mineralized tissues. Although crowned with success, this approach has been restricted so far to a limited set of key-organisms, such as the purple sea urchin Strongylocentrotus purpuratus, the pearl oyster or the abalone, leaving in the shadow non-model organisms. As a consequence, it is still unknown to what extent the calcifying repertoire varies, from group to group, at high (phylum, class), median (order, family) or low (genus, species) taxonomic rank. The present paper shows the first biochemical and proteomic characterization of the test matrix of the Mediterranean black sea urchin Arbacia lixula (Arbacioida). Our work suggests that the skeletal repertoire of A. lixula exhibits some similarities but also several differences with that of the few sea urchin species (S. purpuratus, Paracentrotus lividus), for which molecular data are already available. The differences may be attributable to the taxonomic position of the species considered: A. lixula belongs to an order - Arbacioida - that diverged more than one hundred million years ago from the Camarodonta, which includes the two species S. purpuratus and P. lividus. For the echinoid class, we suggest that large-scale proteomic screening should be performed in order to understand which molecular functions related to calcification are conserved and which ones have been co-opted for biomineralization in particular lineages. Copyright © 2014 Elsevier Inc. All rights reserved.

Binocular pattern deprivation interferes with the expression of proteins involved in primary visual cortex maturation in the cat.

PubMed

Laskowska-Macios, Karolina; Nys, Julie; Hu, Tjing-Tjing; Zapasnik, Monika; Van der Perren, Anke; Kossut, Malgorzata; Burnat, Kalina; Arckens, Lutgarde

2015-08-14

Binocular pattern deprivation from eye opening (early BD) delays the maturation of the primary visual cortex. This delay is more pronounced for the peripheral than the central visual field representation within area 17, particularly between the age of 2 and 4 months [Laskowska-Macios, Cereb Cortex, 2014]. In this study, we probed for related dynamic changes in the cortical proteome. We introduced age, cortical region and BD as principal variables in a 2-D DIGE screen of area 17. In this way we explored the potential of BD-related protein expression changes between central and peripheral area 17 of 2- and 4-month-old BD (2BD, 4BD) kittens as a valid parameter towards the identification of brain maturation-related molecular processes. Consistent with the maturation delay, distinct developmental protein expression changes observed for normal kittens were postponed by BD, especially in the peripheral region. These BD-induced proteomic changes suggest a negative regulation of neurite outgrowth, synaptic transmission and clathrin-mediated endocytosis, thereby implicating these processes in normal experience-induced visual cortex maturation. Verification of the expression of proteins from each of the biological processes via Western analysis disclosed that some of the transient proteomic changes correlate to the distinct behavioral outcome in adult life, depending on timing and duration of the BD period [Neuroscience 2013;255:99-109]. Taken together, the plasticity potential to recover from BD, in relation to ensuing restoration of normal visual input, appears to rely on specific protein expression changes and cellular processes induced by the loss of pattern vision in early life.

Web Extensible Display Manager

DOE Office of Scientific and Technical Information (OSTI.GOV)

Slominski, Ryan; Larrieu, Theodore L.

Jefferson Lab's Web Extensible Display Manager (WEDM) allows staff to access EDM control system screens from a web browser in remote offices and from mobile devices. Native browser technologies are leveraged to avoid installing and managing software on remote clients such as browser plugins, tunnel applications, or an EDM environment. Since standard network ports are used firewall exceptions are minimized. To avoid security concerns from remote users modifying a control system, WEDM exposes read-only access and basic web authentication can be used to further restrict access. Updates of monitored EPICS channels are delivered via a Web Socket using a webmore » gateway. The software translates EDM description files (denoted with the edl suffix) to HTML with Scalable Vector Graphics (SVG) following the EDM's edl file vector drawing rules to create faithful screen renderings. The WEDM server parses edl files and creates the HTML equivalent in real-time allowing existing screens to work without modification. Alternatively, the familiar drag and drop EDM screen creation tool can be used to create optimized screens sized specifically for smart phones and then rendered by WEDM.« less

Ram seminal plasma proteome and its impact on liquid preservation of spermatozoa.

PubMed

Soleilhavoup, C; Tsikis, G; Labas, V; Harichaux, G; Kohnke, P L; Dacheux, J L; Guérin, Y; Gatti, J L; de Graaf, S P; Druart, X

2014-09-23

Seminal plasma is composed of secretions from the epididymis and the accessory sex glands and plays a critical role in the fertilising ability of spermatozoa. In rams, analysis of seminal plasma by GeLC-MS/MS has allowed the identification of more than 700 proteins, including a high abundance of Binder of Sperm family proteins (BSP1, BSP5, SPADH1, SPADH2), the spermadhesin family (bodhesin2), lactoferrin and newly identified proteins like UPF0762 (C6orf58 gene). When spermatogenesis was stopped by scrotal insulation, changes in the proteome profile revealed the sperm origin of 40 seminal proteins, such as glycolysis pathway enzymes, the chaperonin containing TCP1 (CCT) complex and the 26S proteasome complex. Sperm mobility after liquid preservation (24h in milk at 15°C) is male dependent and can be correlated to differences in the seminal plasma proteome, detected by spectral counting. The negative association of zinc alpha-2 glycoprotein (ZAG) with semen preservation was confirmed by the use of recombinant human ZAG, which induced an increase in mobility of fresh sperm, but then decreased sperm mobility after 24h of incubation. Several sperm membrane proteins interacting with the cytoskeleton, glycolysis enzymes and sperm-associated proteins involved in capacitation correlated with better liquid storage and can be considered as seminal biomarkers of sperm preservation. Extensive analysis of the ram seminal plasma proteome reveals a complex and diverse protein composition. This composition varies between males with different sperm preservation abilities. Several proteins were shown to originate from the spermatozoa and positively correlate with sperm liquid preservation, indicating that these proteins can be traced as sperm biomarkers within the seminal plasma. The zinc alpha-2 glycoprotein (ZAG) was found to have a biphasic effect on sperm mobility, with a short-term stimulation followed by a long-term exhaustion of sperm mobility after a 24h preservation period. Copyright © 2014 Elsevier B.V. All rights reserved.

Universal Solid-phase Reversible Sample-Prep for Concurrent Proteome and N-glycome Characterization

PubMed Central

Zhou, Hui; Morley, Samantha; Kostel, Stephen; Freeman, Michael R.; Joshi, Vivek; Brewster, David; Lee, Richard S.

2017-01-01

SUMMARY We describe a novel Solid-phase Reversible Sample-Prep (SRS) platform, which enables rapid sample preparation for concurrent proteome and N-glycome characterization by mass spectrometry. SRS utilizes a uniquely functionalized, silica-based bead that has strong affinity toward proteins with minimal-to-no affinity for peptides and other small molecules. By leveraging the inherent size difference between, SRS permits high-capacity binding of proteins, rapid removal of small molecules (detergents, metabolites, salts, etc.), extensive manipulation including enzymatic and chemical treatments on beads-bound proteins, and easy recovery of N-glycans and peptides. The efficacy of SRS was evaluated in a wide range of biological samples including single glycoprotein, whole cell lysate, murine tissues, and human urine. To further demonstrate the SRS platform, we coupled a quantitative strategy to SRS to investigate the differences between DU145 prostate cancer cells and its DIAPH3-silenced counterpart. Our previous studies suggested that DIAPH3 silencing in DU145 prostate cancer cells induced transition to an amoeboid phenotype that correlated with tumor progression and metastasis. In this analysis we identified distinct proteomic and N-glycomic alterations between the two cells. Intriguingly, a metastasis-associated tyrosine kinase receptor ephrin-type-A receptor (EPHA2) was highly upregulated in DIAPH3-silenced cells, indicating underling connection between EPHA2 and DIAPH3. Moreover, distinct alterations in the N-glycome were identified, suggesting a cross-link between DIAPH3 and glycosyltransferase networks. Overall, SRS is an enabling universal sample preparation strategy that is not size limited and has the capability to efficiently prepare and clean peptides and N-glycans concurrently from nearly all sample types. Conceptually, SRS can be utilized for the analysis of other posttranslational modifications, and the unique surface chemistry can be further transformed for high-throughput automation. The technical simplicity, robustness, and modularity of SRS make it a highly promising technology with great potential in proteomic-based research. PMID:26791391

Overgrazing induces alterations in the hepatic proteome of sheep (Ovis aries): an iTRAQ-based quantitative proteomic analysis.

PubMed

Ren, Weibo; Hou, Xiangyang; Wang, Yuqing; Badgery, Warwick; Li, Xiliang; Ding, Yong; Guo, Huiqin; Wu, Zinian; Hu, Ningning; Kong, Lingqi; Chang, Chun; Jiang, Chao; Zhang, Jize

2016-01-01

The degradation of the steppe of Inner Mongolia, due to overgrazing, has resulted in ecosystem damage as well as extensive reductions in sheep production. The growth performance of sheep is greatly reduced because of overgrazing, which triggers massive economic losses every year. The liver is an essential organ that has very important roles in multiple functions, such as nutrient metabolism, immunity and others, which are closely related to animal growth. However, to our knowledge, no detailed studies have evaluated hepatic metabolism adaption in sheep due to overgrazing. The molecular mechanisms that underlie these effects remain unclear. In the present study, our group applied isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic analysis to investigate changes in the protein profiles of sheep hepatic tissues when nutrition was reduced due to overgrazing (12.0 sheep/ha), with the goal of characterizing the molecular mechanisms of hepatic metabolism adaption in sheep in an overgrazing condition. The body weight daily gain of sheep was greatly decreased due to overgrazing. Overall, 41 proteins were found to be differentially abundant in the hepatic tissue between a light grazing group and an overgrazing group. Most of the differentially expressed proteins identified are involved in protein metabolism, transcriptional and translational regulation, and immune response. In particular, the altered abundance of kynureninase (KYNU) and HAL (histidine ammonia-lyase) involved in protein metabolic function, integrated with the changes of serum levels of blood urea nitrogen (BUN) and glucose (GLU), suggest that overgrazing triggers a shift in energy resources from carbohydrates to proteins, causing poorer nitrogen utilization efficiency. Altogether, these results suggest that the reductions in animal growth induced by overgrazing are associated with liver proteomic changes, especially the proteins involved in nitrogen compounds metabolism and immunity. This provides new information that can be used for nutritional supplementation to improve the growth performance of sheep in an overgrazing condition.

Differential proteomics reveals the hallmarks of seed development in common bean (Phaseolus vulgaris L.).

PubMed

Parreira, J R; Bouraada, J; Fitzpatrick, M A; Silvestre, S; Bernardes da Silva, A; Marques da Silva, J; Almeida, A M; Fevereiro, P; Altelaar, A F M; Araújo, S S

2016-06-30

Common bean (Phaseolus vulgaris L.) is one of the most consumed staple foods worldwide. Little is known about the molecular mechanisms controlling seed development. This study aims to comprehensively describe proteome dynamics during seed development of common bean. A high-throughput gel-free proteomics approach (LC-MS/MS) was conducted on seeds at 10, 20, 30 and 40days after anthesis, spanning from late embryogenesis until desiccation. Of the 418 differentially accumulated proteins identified, 255 were characterized, most belonging to protein metabolism. An accumulation of proteins belonging to the MapMan functional categories of "protein", "glycolysis", "TCA", "DNA", "RNA", "cell" and "stress" were found at early seed development stages, reflecting an extensive metabolic activity. In the mid stages, accumulation of storage, signaling, starch synthesis and cell wall-related proteins stood out. In the later stages, an increase in proteins related to redox, protein degradation/modification/folding and nucleic acid metabolisms reflect that seed desiccation-resistance mechanisms were activated. Our study unveils new clues to understand the regulation of seed development mediated by post-translational modifications and maintenance of genome integrity. This knowledge enhances the understanding on seed development molecular mechanisms that may be used in the design and selection of common bean seeds with desired quality traits. Common bean (P. vulgaris) is an important source of proteins and carbohydrates worldwide. Despite the agronomic and economic importance of this pulse, knowledge on common bean seed development is limited. Herein, a gel-free high throughput methodology was used to describe the proteome changes during P. vulgaris seed development. Data obtained will enhance the knowledge on the molecular mechanisms controlling this grain legume seed development and may be used in the design and selection of common bean seeds with desired quality traits. Results may be extrapolated to other pulses. Copyright © 2016 Elsevier B.V. All rights reserved.

A comprehensive proteomics and genomics analysis reveals novel transmembrane proteins in human platelets and mouse megakaryocytes including G6b-B, a novel ITIM protein

PubMed Central

Senis, Yotis A.; Tomlinson, Michael G.; García, Ángel; Dumon, Stephanie; Heath, Victoria L.; Herbert, John; Cobbold, Stephen P.; Spalton, Jennifer C.; Ayman, Sinem; Antrobus, Robin; Zitzmann, Nicole; Bicknell, Roy; Frampton, Jon; Authi, Kalwant; Martin, Ashley; Wakelam, Michael J.O.; Watson, Stephen P.

2007-01-01

Summary The platelet surface is poorly characterized due to the low abundance of many membrane proteins and the lack of specialist tools for their investigation. In this study we have identified novel human platelet and mouse megakaryocyte membrane proteins using specialist proteomic and genomic approaches. Three separate methods were used to enrich platelet surface proteins prior to identification by liquid chromatography and tandem mass spectrometry: lectin affinity chromatography; biotin/NeutrAvidin affinity chromatography; and free flow electrophoresis. Many known, abundant platelet surface transmembrane proteins and several novel proteins were identified using each receptor enrichment strategy. In total, two or more unique peptides were identified for 46, 68 and 22 surface membrane, intracellular membrane and membrane proteins of unknown sub-cellular localization, respectively. The majority of these were single transmembrane proteins. To complement the proteomic studies, we analysed the transcriptome of a highly purified preparation of mature primary mouse megakaryocytes using serial analysis of gene expression in view of the increasing importance of mutant mouse models in establishing protein function in platelets. This approach identified all of the major classes of platelet transmembrane receptors, including multi-transmembrane proteins. Strikingly, 17 of the 25 most megakaryocyte-specific genes (relative to 30 other SAGE libraries) were transmembrane proteins, illustrating the unique nature of the megakaryocyte/platelet surface. The list of novel plasma membrane proteins identified using proteomics includes the immunoglobulin superfamily member G6b, which undergoes extensive alternate splicing. Specific antibodies were used to demonstrate expression of the G6b-B isoform, which contains an immunoreceptor tyrosine-based inhibition motif. G6b-B undergoes tyrosine phosphorylation and association with the SH2-containing phosphatase, SHP-1, in stimulated platelets suggesting that it may play a novel role in limiting platelet activation. PMID:17186946

A Comparative Proteomic Analysis of the Simple Amino Acid Repeat Distributions in Plasmodia Reveals Lineage Specific Amino Acid Selection

PubMed Central

Dalby, Andrew R.

2009-01-01

Background Microsatellites have been used extensively in the field of comparative genomics. By studying microsatellites in coding regions we have a simple model of how genotypic changes undergo selection as they are directly expressed in the phenotype as altered proteins. The simplest of these tandem repeats in coding regions are the tri-nucleotide repeats which produce a repeat of a single amino acid when translated into proteins. Tri-nucleotide repeats are often disease associated, and are also known to be unstable to both expansion and contraction. This makes them sensitive markers for studying proteome evolution, in closely related species. Results The evolutionary history of the family of malarial causing parasites Plasmodia is complex because of the life-cycle of the organism, where it interacts with a number of different hosts and goes through a series of tissue specific stages. This study shows that the divergence between the primate and rodent malarial parasites has resulted in a lineage specific change in the simple amino acid repeat distribution that is correlated to A–T content. The paper also shows that this altered use of amino acids in SAARs is consistent with the repeat distributions being under selective pressure. Conclusions The study shows that simple amino acid repeat distributions can be used to group related species and to examine their phylogenetic relationships. This study also shows that an outgroup species with a similar A–T content can be distinguished based only on the amino acid usage in repeats, and suggest that this might be a useful feature for proteome clustering. The lineage specific use of amino acids in repeat regions suggests that comparative studies of SAAR distributions between proteomes gives an insight into the mechanisms of expansion and the selective pressures acting on the organism. PMID:19597555

Identification and Characterization of Soluble Factors Involved in Delayed Effects of Low Dose Radiation. Final report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baulch, Janet

2013-09-11

This is a 'glue grant' that was part of a DOE Low Dose project entitled 'Identification and Characterization of Soluble Factors Involved in Delayed Effects of Low Dose Radiation'. This collaborative program has involved Drs. David L. Springer from Pacific Northwest National Laboratory (PNNL), John H. Miller from Washington State University, Tri-cities (WSU) and William F. Morgan then from the University of Maryland, Baltimore (UMB). In July 2008, Dr. Morgan moved to PNNL and Dr. Janet E. Baulch became PI for this project at University of Maryland. In November of 2008, a one year extension with no new funds wasmore » requested to complete the proteomic analyses. The project stemmed from studies in the Morgan laboratory demonstrating that genomically unstable cells secret a soluble factor or factors into the culture medium, that cause cytogenetic aberrations and apoptosis in normal parental GM10115 cells. The purpose of this project was to identify the death inducing effect (DIE) factor or factors, estimate their relative abundance, identify the cell signaling pathways involved and finally recapitulate DIE in normal cells by exogenous manipulation of putative DIE factors in culture medium. As reported in detail in the previous progress report, analysis of culture medium from the parental cell line, and stable and unstable clones demonstrated inconsistent proteomic profiles as relate to candidate DIE factors. While the proposed proteomic analyses did not provide information that would allow DIE factors to be identified, the analyses provided another important set of observations. Proteomic analysis suggested that proteins associated with the cellular response to oxidative stress and mitochondrial function were elevated in the medium from unstable clones in a manner consistent with mitochondrial dysfunction. These findings correlate with previous studies of these clones that demonstrated functional differences between the mitochondria of stable and unstable clones. These mitochondrial abnormalities in the unstable clones contributes to oxidative stress.« less

Analysis of human serum phosphopeptidome by a focused database searching strategy.

PubMed

Zhu, Jun; Wang, Fangjun; Cheng, Kai; Song, Chunxia; Qin, Hongqiang; Hu, Lianghai; Figeys, Daniel; Ye, Mingliang; Zou, Hanfa

2013-01-14

As human serum is an important source for early diagnosis of many serious diseases, analysis of serum proteome and peptidome has been extensively performed. However, the serum phosphopeptidome was less explored probably because the effective method for database searching is lacking. Conventional database searching strategy always uses the whole proteome database, which is very time-consuming for phosphopeptidome search due to the huge searching space resulted from the high redundancy of the database and the setting of dynamic modifications during searching. In this work, a focused database searching strategy using an in-house collected human serum pro-peptidome target/decoy database (HuSPep) was established. It was found that the searching time was significantly decreased without compromising the identification sensitivity. By combining size-selective Ti (IV)-MCM-41 enrichment, RP-RP off-line separation, and complementary CID and ETD fragmentation with the new searching strategy, 143 unique endogenous phosphopeptides and 133 phosphorylation sites (109 novel sites) were identified from human serum with high reliability. Copyright © 2012 Elsevier B.V. All rights reserved.

Significant expansion of exon-bordering protein domains during animal proteome evolution

PubMed Central

Liu, Mingyi; Walch, Heiko; Wu, Shaoping; Grigoriev, Andrei

2005-01-01

We present evidence of remarkable genome-wide mobility and evolutionary expansion for a class of protein domains whose borders locate close to the borders of their encoding exons. These exon-bordering domains are more numerous and widely distributed in the human genome than other domains. They also co-occur with more diverse domains to form a larger variety of domain architectures in human proteins. A systematic comparison of nine animal genomes from nematodes to mammals revealed that exon-bordering domains expanded faster than other protein domains in both abundance and distribution, as well as the diversity of co-occurring domains and the domain architectures of harboring proteins. Furthermore, exon-bordering domains exhibited a particularly strong preference for class 1-1 intron phase. Our findings suggest that exon-bordering domains were amplified and interchanged within a genome more often and/or more successfully than other domains during evolution, probably the result of extensive exon shuffling and gene duplication events. The diverse biological functions of these domains underscore the important role they play in the expansion and diversification of animal proteomes. PMID:15640447

Biomarkers for ragwort poisoning in horses: identification of protein targets

PubMed Central

Moore, Rowan E; Knottenbelt, Derek; Matthews, Jacqueline B; Beynon, Robert J; Whitfield, Phillip D

2008-01-01

Background Ingestion of the poisonous weed ragwort (Senecio jacobea) by horses leads to irreversible liver damage. The principal toxins of ragwort are the pyrrolizidine alkaloids that are rapidly metabolised to highly reactive and cytotoxic pyrroles, which can escape into the circulation and bind to proteins. In this study a non-invasive in vitro model system has been developed to investigate whether pyrrole toxins induce specific modifications of equine blood proteins that are detectable by proteomic methods. Results One dimensional gel electrophoresis revealed a significant alteration in the equine plasma protein profile following pyrrole exposure and the formation of a high molecular weight protein aggregate. Using mass spectrometry and confirmation by western blotting the major components of this aggregate were identified as fibrinogen, serum albumin and transferrin. Conclusion These findings demonstrate that pyrrolic metabolites can modify equine plasma proteins. The high molecular weight aggregate may result from extensive inter- and intra-molecular cross-linking of fibrinogen with the pyrrole. This model has the potential to form the basis of a novel proteomic strategy aimed at identifying surrogate protein biomarkers of ragwort exposure in horses and other livestock. PMID:18691403

Metabolomics and Proteomics of Brassica napus Guard Cells in Response to Low CO2

PubMed Central

Geng, Sisi; Yu, Bing; Zhu, Ning; Dufresne, Craig; Chen, Sixue

2017-01-01

Stomatal guard cell response to various stimuli is an important process that balances plant carbon dioxide (CO2) uptake and water transpiration. Elevated CO2 induces stomatal closure, while low CO2 promotes stomatal opening. The signaling process of elevated CO2 induced stomatal closure has been extensively studied in recent years. However, the mechanism of low CO2 induced stomatal opening is not fully understood. Here we report metabolomic and proteomic responses of Brassica napus guard cells to low CO2 using hyphenated mass spectrometry technologies. A total of 411 metabolites and 1397 proteins were quantified in a time-course study of low CO2 effects. Metabolites and proteins that exhibited significant changes are overrepresented in fatty acid metabolism, starch and sucrose metabolism, glycolysis and redox regulation. Concomitantly, multiple hormones that promote stomatal opening increased in response to low CO2. Interestingly, jasmonic acid precursors were diverted to a branch pathway of traumatic acid biosynthesis. These results indicate that the low CO2 response is mediated by a complex crosstalk between different phytohormones. PMID:28791296

Integrated Proteomics and Metabolomics Suggests Symbiotic Metabolism and Multimodal Regulation in a Fungal-Endobacterial System: Symbiotic Metabolism and Multimodal Regulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Zhou; Yao, Qiuming; Dearth, Stephen P.

Many plant-associated fungi host endosymbiotic endobacteria with reduced genomes. While endobacteria play important roles in these tri-partite plant-fungal-endobacterial systems, the active physiology of fungal endobacteria has not been characterized extensively by systems biology approaches. Here in this paper, we use integrated proteomics and metabolomics to characterize the relationship between the endobacterium Mycoavidus sp. and the root-associated fungus Mortierella elongata. In nitrogen-poor media, M. elongata had decreased growth but hosted a large and growing endobacterial population. The active endobacterium likely extracted malate from the fungal host as the primary carbon substrate for energy production and biosynthesis of phospho-sugars, nucleobases, peptidoglycan, andmore » some amino acids. The endobacterium obtained nitrogen by importing a variety of nitrogen-containing compounds. Further, nitrogen limitation significantly perturbed the carbon and nitrogen flows in the fungal metabolic network. M. elongata regulated many pathways by concordant changes on enzyme abundances, post-translational modifications, reactant concentrations, and allosteric effectors. Lastly, such multimodal regulations may be a general mechanism for metabolic modulation.« less

jmzIdentML API: A Java interface to the mzIdentML standard for peptide and protein identification data.

PubMed

Reisinger, Florian; Krishna, Ritesh; Ghali, Fawaz; Ríos, Daniel; Hermjakob, Henning; Vizcaíno, Juan Antonio; Jones, Andrew R

2012-03-01

We present a Java application programming interface (API), jmzIdentML, for the Human Proteome Organisation (HUPO) Proteomics Standards Initiative (PSI) mzIdentML standard for peptide and protein identification data. The API combines the power of Java Architecture of XML Binding (JAXB) and an XPath-based random-access indexer to allow a fast and efficient mapping of extensible markup language (XML) elements to Java objects. The internal references in the mzIdentML files are resolved in an on-demand manner, where the whole file is accessed as a random-access swap file, and only the relevant piece of XMLis selected for mapping to its corresponding Java object. The APIis highly efficient in its memory usage and can handle files of arbitrary sizes. The APIfollows the official release of the mzIdentML (version 1.1) specifications and is available in the public domain under a permissive licence at http://www.code.google.com/p/jmzidentml/. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Metabolomics and Proteomics of Brassica napus Guard Cells in Response to Low CO2.

PubMed

Geng, Sisi; Yu, Bing; Zhu, Ning; Dufresne, Craig; Chen, Sixue

2017-01-01

Stomatal guard cell response to various stimuli is an important process that balances plant carbon dioxide (CO 2 ) uptake and water transpiration. Elevated CO 2 induces stomatal closure, while low CO 2 promotes stomatal opening. The signaling process of elevated CO 2 induced stomatal closure has been extensively studied in recent years. However, the mechanism of low CO 2 induced stomatal opening is not fully understood. Here we report metabolomic and proteomic responses of Brassica napus guard cells to low CO 2 using hyphenated mass spectrometry technologies. A total of 411 metabolites and 1397 proteins were quantified in a time-course study of low CO 2 effects. Metabolites and proteins that exhibited significant changes are overrepresented in fatty acid metabolism, starch and sucrose metabolism, glycolysis and redox regulation. Concomitantly, multiple hormones that promote stomatal opening increased in response to low CO 2 . Interestingly, jasmonic acid precursors were diverted to a branch pathway of traumatic acid biosynthesis. These results indicate that the low CO 2 response is mediated by a complex crosstalk between different phytohormones.

Defining the Protein–Protein Interaction Network of the Human Hippo Pathway*

PubMed Central

Wang, Wenqi; Li, Xu; Huang, Jun; Feng, Lin; Dolinta, Keithlee G.; Chen, Junjie

2014-01-01

The Hippo pathway, which is conserved from Drosophila to mammals, has been recognized as a tumor suppressor signaling pathway governing cell proliferation and apoptosis, two key events involved in organ size control and tumorigenesis. Although several upstream regulators, the conserved kinase cascade and key downstream effectors including nuclear transcriptional factors have been defined, the global organization of this signaling pathway is not been fully understood. Thus, we conducted a proteomic analysis of human Hippo pathway, which revealed the involvement of an extensive protein–protein interaction network in this pathway. The mass spectrometry data were deposited to ProteomeXchange with identifier PXD000415. Our data suggest that 550 interactions within 343 unique protein components constitute the central protein–protein interaction landscape of human Hippo pathway. Our study provides a glimpse into the global organization of Hippo pathway, reveals previously unknown interactions within this pathway, and uncovers new potential components involved in the regulation of this pathway. Understanding these interactions will help us further dissect the Hippo signaling-pathway and extend our knowledge of organ size control. PMID:24126142

Quantitative comparison of a human cancer cell surface proteome between interphase and mitosis.

PubMed

Özlü, Nurhan; Qureshi, Mohammad H; Toyoda, Yusuke; Renard, Bernhard Y; Mollaoglu, Gürkan; Özkan, Nazlı E; Bulbul, Selda; Poser, Ina; Timm, Wiebke; Hyman, Anthony A; Mitchison, Timothy J; Steen, Judith A

2015-01-13

The cell surface is the cellular compartment responsible for communication with the environment. The interior of mammalian cells undergoes dramatic reorganization when cells enter mitosis. These changes are triggered by activation of the CDK1 kinase and have been studied extensively. In contrast, very little is known of the cell surface changes during cell division. We undertook a quantitative proteomic comparison of cell surface-exposed proteins in human cancer cells that were tightly synchronized in mitosis or interphase. Six hundred and twenty-eight surface and surface-associated proteins in HeLa cells were identified; of these, 27 were significantly enriched at the cell surface in mitosis and 37 in interphase. Using imaging techniques, we confirmed the mitosis-selective cell surface localization of protocadherin PCDH7, a member of a family with anti-adhesive roles in embryos. We show that PCDH7 is required for development of full mitotic rounding pressure at the onset of mitosis. Our analysis provided basic information on how cell cycle progression affects the cell surface. It also provides potential pharmacodynamic biomarkers for anti-mitotic cancer chemotherapy. © 2014 The Authors.

Quantitative comparison of a human cancer cell surface proteome between interphase and mitosis

PubMed Central

Özlü, Nurhan; Qureshi, Mohammad H; Toyoda, Yusuke; Renard, Bernhard Y; Mollaoglu, Gürkan; Özkan, Nazlı E; Bulbul, Selda; Poser, Ina; Timm, Wiebke; Hyman, Anthony A; Mitchison, Timothy J; Steen, Judith A

2015-01-01

The cell surface is the cellular compartment responsible for communication with the environment. The interior of mammalian cells undergoes dramatic reorganization when cells enter mitosis. These changes are triggered by activation of the CDK1 kinase and have been studied extensively. In contrast, very little is known of the cell surface changes during cell division. We undertook a quantitative proteomic comparison of cell surface-exposed proteins in human cancer cells that were tightly synchronized in mitosis or interphase. Six hundred and twenty-eight surface and surface-associated proteins in HeLa cells were identified; of these, 27 were significantly enriched at the cell surface in mitosis and 37 in interphase. Using imaging techniques, we confirmed the mitosis-selective cell surface localization of protocadherin PCDH7, a member of a family with anti-adhesive roles in embryos. We show that PCDH7 is required for development of full mitotic rounding pressure at the onset of mitosis. Our analysis provided basic information on how cell cycle progression affects the cell surface. It also provides potential pharmacodynamic biomarkers for anti-mitotic cancer chemotherapy. PMID:25476450

Global functional atlas of Escherichia coli encompassing previously uncharacterized proteins.

PubMed

Hu, Pingzhao; Janga, Sarath Chandra; Babu, Mohan; Díaz-Mejía, J Javier; Butland, Gareth; Yang, Wenhong; Pogoutse, Oxana; Guo, Xinghua; Phanse, Sadhna; Wong, Peter; Chandran, Shamanta; Christopoulos, Constantine; Nazarians-Armavil, Anaies; Nasseri, Negin Karimi; Musso, Gabriel; Ali, Mehrab; Nazemof, Nazila; Eroukova, Veronika; Golshani, Ashkan; Paccanaro, Alberto; Greenblatt, Jack F; Moreno-Hagelsieb, Gabriel; Emili, Andrew

2009-04-28

One-third of the 4,225 protein-coding genes of Escherichia coli K-12 remain functionally unannotated (orphans). Many map to distant clades such as Archaea, suggesting involvement in basic prokaryotic traits, whereas others appear restricted to E. coli, including pathogenic strains. To elucidate the orphans' biological roles, we performed an extensive proteomic survey using affinity-tagged E. coli strains and generated comprehensive genomic context inferences to derive a high-confidence compendium for virtually the entire proteome consisting of 5,993 putative physical interactions and 74,776 putative functional associations, most of which are novel. Clustering of the respective probabilistic networks revealed putative orphan membership in discrete multiprotein complexes and functional modules together with annotated gene products, whereas a machine-learning strategy based on network integration implicated the orphans in specific biological processes. We provide additional experimental evidence supporting orphan participation in protein synthesis, amino acid metabolism, biofilm formation, motility, and assembly of the bacterial cell envelope. This resource provides a "systems-wide" functional blueprint of a model microbe, with insights into the biological and evolutionary significance of previously uncharacterized proteins.

Integrated Proteomics and Metabolomics Suggests Symbiotic Metabolism and Multimodal Regulation in a Fungal-Endobacterial System: Symbiotic Metabolism and Multimodal Regulation

DOE PAGES

Li, Zhou; Yao, Qiuming; Dearth, Stephen P.; ...

2016-11-21

Many plant-associated fungi host endosymbiotic endobacteria with reduced genomes. While endobacteria play important roles in these tri-partite plant-fungal-endobacterial systems, the active physiology of fungal endobacteria has not been characterized extensively by systems biology approaches. Here in this paper, we use integrated proteomics and metabolomics to characterize the relationship between the endobacterium Mycoavidus sp. and the root-associated fungus Mortierella elongata. In nitrogen-poor media, M. elongata had decreased growth but hosted a large and growing endobacterial population. The active endobacterium likely extracted malate from the fungal host as the primary carbon substrate for energy production and biosynthesis of phospho-sugars, nucleobases, peptidoglycan, andmore » some amino acids. The endobacterium obtained nitrogen by importing a variety of nitrogen-containing compounds. Further, nitrogen limitation significantly perturbed the carbon and nitrogen flows in the fungal metabolic network. M. elongata regulated many pathways by concordant changes on enzyme abundances, post-translational modifications, reactant concentrations, and allosteric effectors. Lastly, such multimodal regulations may be a general mechanism for metabolic modulation.« less

Development of an Enhanced Metaproteomic Approach for Deepening the Microbiome Characterization of the Human Infant Gut

PubMed Central

2015-01-01

The establishment of early life microbiota in the human infant gut is highly variable and plays a crucial role in host nutrient availability/uptake and maturation of immunity. Although high-performance mass spectrometry (MS)-based metaproteomics is a powerful method for the functional characterization of complex microbial communities, the acquisition of comprehensive metaproteomic information in human fecal samples is inhibited by the presence of abundant human proteins. To alleviate this restriction, we have designed a novel metaproteomic strategy based on double filtering (DF) the raw samples, a method that fractionates microbial from human cells to enhance microbial protein identification and characterization in complex fecal samples from healthy premature infants. This method dramatically improved the overall depth of infant gut proteome measurement, with an increase in the number of identified low-abundance proteins and a greater than 2-fold improvement in microbial protein identification and quantification. This enhancement of proteome measurement depth enabled a more extensive microbiome comparison between infants by not only increasing the confidence of identified microbial functional categories but also revealing previously undetected categories. PMID:25350865