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Sample records for gender-specific proteomic profiling

  1. Brugia malayi Excreted/Secreted Proteins at the Host/Parasite Interface: Stage- and Gender-Specific Proteomic Profiling

    PubMed Central

    Bennuru, Sasisekhar; Semnani, Roshanak; Meng, Zhaojing; Ribeiro, Jose M. C.; Veenstra, Timothy D.; Nutman, Thomas B.

    2009-01-01

    Relatively little is known about the filarial proteins that interact with the human host. Although the filarial genome has recently been completed, protein profiles have been limited to only a few recombinants or purified proteins of interest. Here, we describe a large-scale proteomic analysis using microcapillary reverse-phase liquid chromatography-tandem-mass spectrometry to identify the excretory-secretory (ES) products of the L3, L3 to L4 molting ES, adult male, adult female, and microfilarial stages of the filarial parasite Brugia malayi. The analysis of the ES products from adult male, adult female, microfilariae (Mf), L3, and molting L3 larvae identified 852 proteins. Annotation suggests that the functional and component distribution was very similar across each of the stages studied; however, the Mf contributed a higher proportion to the total number of identified proteins than the other stages. Of the 852 proteins identified in the ES, only 229 had previous confirmatory expressed sequence tags (ESTs) in the available databases. Moreover, this analysis was able to confirm the presence of 274 “hypothetical” proteins inferred from gene prediction algorithms applied to the B. malayi (Bm) genome. Not surprisingly, the majority (160/274) of these “hypothetical” proteins were predicted to be secreted by Signal IP and/or SecretomeP 2.0 analysis. Of major interest is the abundance of previously characterized immunomodulatory proteins such as ES-62 (leucyl aminopeptidase), MIF-1, SERPIN, glutathione peroxidase, and galectin in the ES of microfilariae (and Mf-containing adult females) compared to the adult males. In addition, searching the ES protein spectra against the Wolbachia database resulted in the identification of 90 Wolbachia-specific proteins, most of which were metabolic enzymes that have not been shown to be immunogenic. This proteomic analysis extends our knowledge of the ES and provides insight into the host–parasite interaction. PMID:19352421

  2. Stage- and Gender-Specific Proteomic Analysis of Brugia malayi Excretory-Secretory Products

    PubMed Central

    Moreno, Yovany; Geary, Timothy G.

    2008-01-01

    Introduction While we lack a complete understanding of the molecular mechanisms by which parasites establish and achieve protection from host immune responses, it is accepted that many of these processes are mediated by products, primarily proteins, released from the parasite. Parasitic nematodes occur in different life stages and anatomical compartments within the host. Little is known about the composition and variability of products released at different developmental stages and their contribution to parasite survival and progression of the infection. Methodology/Principal Findings To gain a deeper understanding on these aspects, we collected and analyzed through 1D-SDS PAGE and LC-MS/MS the Excretory-Secretory Products (ESP) of adult female, adult male and microfilariae of the filarial nematode Brugia malayi, one of the etiological agents of human lymphatic filariasis. This proteomic analysis led to the identification of 228 proteins. The list includes 76 proteins with unknown function as well as also proteins with potential immunoregulatory properties, such as protease inhibitors, cytokine homologues and carbohydrate-binding proteins. Larval and adult ESP differed in composition. Only 32 proteins were shared between all three stages/genders. Consistent with this observation, different gene ontology profiles were associated with the different ESP. Conclusions/Significance A comparative analysis of the proteins released in vitro by different forms of a parasitic nematode dwelling in the same host is presented. The catalog of secreted proteins reflects different stage- and gender-specific related processes and different strategies of immune evasion, providing valuable insights on the contribution of each form of the parasite for establishing the host–parasite interaction. PMID:18958170

  3. Proteome-wide alterations on adipose tissue from obese patients as age-, diabetes- and gender-specific hallmarks.

    PubMed

    Gómez-Serrano, María; Camafeita, Emilio; García-Santos, Eva; López, Juan A; Rubio, Miguel A; Sánchez-Pernaute, Andrés; Torres, Antonio; Vázquez, Jesús; Peral, Belén

    2016-01-01

    Obesity is a main global health issue and an outstanding cause of morbidity and mortality predisposing to type 2 diabetes (T2DM) and cardiovascular diseases. Huge research efforts focused on gene expression, cellular signalling and metabolism in obesity have improved our understanding of these disorders; nevertheless, to bridge the gap between the regulation of gene expression and changes in signalling/metabolism, protein levels must be assessed. We have extensively analysed visceral adipose tissue from age-, T2DM- and gender-matched obese patients using high-throughput proteomics and systems biology methods to identify new biomarkers for the onset of T2DM in obesity, as well as to gain insight into the influence of aging and gender in these disorders. About 250 proteins showed significant abundance differences in the age, T2DM and gender comparisons. In diabetic patients, remarkable gender-specific hallmarks were discovered regarding redox status, immune response and adipose tissue accumulation. Both aging and T2DM processes were associated with mitochondrial remodelling, albeit through well-differentiated proteome changes. Systems biology analysis highlighted mitochondrial proteins that could play a key role in the age-dependent pathophysiology of T2DM. Our findings could serve as a framework for future research in Translational Medicine directed at improving the quality of life of obese patients. PMID:27160966

  4. Proteome-wide alterations on adipose tissue from obese patients as age-, diabetes- and gender-specific hallmarks

    PubMed Central

    Gómez-Serrano, María; Camafeita, Emilio; García-Santos, Eva; López, Juan A.; Rubio, Miguel A.; Sánchez-Pernaute, Andrés; Torres, Antonio; Vázquez, Jesús; Peral, Belén

    2016-01-01

    Obesity is a main global health issue and an outstanding cause of morbidity and mortality predisposing to type 2 diabetes (T2DM) and cardiovascular diseases. Huge research efforts focused on gene expression, cellular signalling and metabolism in obesity have improved our understanding of these disorders; nevertheless, to bridge the gap between the regulation of gene expression and changes in signalling/metabolism, protein levels must be assessed. We have extensively analysed visceral adipose tissue from age-, T2DM- and gender-matched obese patients using high-throughput proteomics and systems biology methods to identify new biomarkers for the onset of T2DM in obesity, as well as to gain insight into the influence of aging and gender in these disorders. About 250 proteins showed significant abundance differences in the age, T2DM and gender comparisons. In diabetic patients, remarkable gender-specific hallmarks were discovered regarding redox status, immune response and adipose tissue accumulation. Both aging and T2DM processes were associated with mitochondrial remodelling, albeit through well-differentiated proteome changes. Systems biology analysis highlighted mitochondrial proteins that could play a key role in the age-dependent pathophysiology of T2DM. Our findings could serve as a framework for future research in Translational Medicine directed at improving the quality of life of obese patients. PMID:27160966

  5. Gender-specific transcriptional profiling of marine medaka (Oryzias melastigma) liver upon BDE-47 exposure.

    PubMed

    Yu, W K; Shi, Y F; Fong, C C; Chen, Y; van de Merwe, J P; Chan, Alice K Y; Wei, F; Bo, J; Ye, R; Au, Doris W T; Wu, Rudolf S S; Yang, M S

    2013-09-01

    Marine medaka (Oryzias melastigma) were exposed to 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) to investigate the gender-specific transcriptional profiles of liver tissue in response to this flame retardant. A cDNA library of O. melastigma was constructed, and 2304 clones were amplified from the library to fabricate a cDNA microarray. Sequences of these genes were assembled into 1800 sequences using Geneious, a bioinformatics software. Corresponding expressed sequence tags were blasted against the National Centre for Biotechnology Information non-redundant database and further classified into various biological categories according to the Gene Ontology project. Male and female three-month-old were fed a diet of BDE-47 contaminated Artemia at low dosage (290.3±172.3ng BDE-47/day) and high dosage (580.5±344.6ng BDE-47/day) for 5 and 21 days, respectively. The transcriptional profiles of O. melastigma liver were then generated by the species-specific cDNA microrarray. The results from microarray analysis suggested very different gene expression patterns between males and females for both BDE-47 exposure-dose and exposure-time, with male livers having stronger gene regulatory responses than female livers. Importantly, our results revealed that in male O. melastigma only, BDE-47 exposure may activate phosphoinositide-3-kinase and mitogen-activated protein kinase, proteins that play importance roles in cell growth, proliferation and survival. PMID:23962555

  6. Proteomic profiling of skeletal muscle plasticity

    PubMed Central

    Ohlendieck, Kay

    2011-01-01

    Summary One of the most striking physiological features of skeletal muscle tissues are their enormous capacity to adapt to changed functional demands. Muscle plasticity has been extensively studied by histological, biochemical, physiological and genetic methods over the last few decades. With the recent emergence of high-throughput and large-scale proteomic techniques, mass spectrometry-based surveys have also been applied to the global analysis of the skeletal muscle protein complement during physiological modifications and pathophysiological alterations. This review outlines and discusses the impact of recent proteomic profiling studies of skeletal muscle transitions, including the effects of chronic electro-stimulation, physical exercise, denervation, disuse atrophy, hypoxia, myotonia, motor neuron disease and age-related fibre type shifting. This includes studies on the human skeletal muscle proteome, animal models of muscle plasticity and major neuromuscular pathologies. The biomedical importance of establishing reliable biomarker signatures for the various molecular and cellular transition phases involved in muscle transformation is critically examined. PMID:23738259

  7. Proteomic profiling of skeletal muscle plasticity.

    PubMed

    Ohlendieck, Kay

    2011-10-01

    One of the most striking physiological features of skeletal muscle tissues are their enormous capacity to adapt to changed functional demands. Muscle plasticity has been extensively studied by histological, biochemical, physiological and genetic methods over the last few decades. With the recent emergence of high-throughput and large-scale proteomic techniques, mass spectrometry-based surveys have also been applied to the global analysis of the skeletal muscle protein complement during physiological modifications and pathophysiological alterations. This review outlines and discusses the impact of recent proteomic profiling studies of skeletal muscle transitions, including the effects of chronic electro-stimulation, physical exercise, denervation, disuse atrophy, hypoxia, myotonia, motor neuron disease and age-related fibre type shifting. This includes studies on the human skeletal muscle proteome, animal models of muscle plasticity and major neuromuscular pathologies. The biomedical importance of establishing reliable biomarker signatures for the various molecular and cellular transition phases involved in muscle transformation is critically examined. PMID:23738259

  8. Shotgun proteome profile of Populus developing xylem

    SciTech Connect

    Kalluri, Udaya C; Hurst, Gregory {Greg} B; Lankford, Patricia K; Ranjan, Priya; Pelletier, Dale A

    2009-01-01

    Understanding the molecular pathways of plant cell wall biosynthesis and remodeling is central to interpreting biological mechanisms underlying plant growth and adaptation as well as leveraging that knowledge towards development of improved bioenergy feedstocks. Here we report the application of shotgun tandem mass spectrometry profiling to the proteome of Populus developing xylem. Additionally, we mined public databases to obtain information in support of subcellular localization, transcript-level expression, and functional categorization of these proteins. Nearly 6000 different proteins were identified from the xylem proteome, with over 4400 proteins identified from one or more unique peptides. In addition to finding protein-level evidence of candidate wall biosynthesis genes from xylem (wood) tissue such as cellulose synthase, phenylalanine ammonia-lyase, and 4-coumarate:CoA ligase, several other potentially new candidate genes in the pathway were discovered. In order to identify low-abundance DNA-regulatory proteins from the developing xylem, a selective nuclear proteome profiling method was developed. Several putative transcription factor and chromatin remodeling proteins were identified using this method, such as LIM and NAC domain transcription factors and CHB3-SWI/SNF-related proteins. Further application of these proteomics methods will enhance understanding not only of cell wall biosynthesis in system biology modeling, but also other plant developmental and physiological pathways.

  9. Global MS-Based Proteomics Drug Profiling.

    PubMed

    Carvalho, Ana Sofia; Matthiesen, Rune

    2016-01-01

    DNA-based technologies such as RNAi, chemical-genetic profiling, or gene expression profiling by DNA microarrays combined with other biochemical methods are established strategies for surveying drug mechanisms. Such approaches can provide mechanistic information on how drugs act and affect cellular pathways. By studying how cancer cells compensate for the drug treatment, novel targets used in a combined treatment can be designed. Furthermore, toxicity effects on cells not targeted can be obtained on a molecular level. For example, drug companies are particularly interested in studying the molecular side effects of drugs in the liver. In addition, experiments with the purpose of elucidating liver toxicity can be studied using samples obtained from animal models exposed to different concentrations of a drug over time. More recently considerable advances in mass spectrometry (MS) technologies and bioinformatics tools allows informative global drug profiling experiments to be performed at a cost comparable to other large-scale technologies such as DNA-based technologies. Moreover, MS-based proteomics provides an additional layer of information on the dynamic regulation of proteins translation and particularly protein degradation. MS-based proteomics approaches combined with other biochemical methods delivers information on regulatory networks, signaling cascades, and metabolic pathways upon drug treatment. Furthermore, MS-based proteomics can provide additional information on single amino acid polymorphisms, protein isoform distribution, posttranslational modifications, and subcellular localization. In this chapter, we will share our experience using MS based proteomics as a pharmacoproteomics strategy to characterize drug mechanisms of action in single drug therapy or in multidrug combination. Finally, the emergence of integrated proteogenomics analysis, such as "The Cancer Genome Atlas" program, opened interesting perspectives to extend this approach to drug target

  10. Distinctive proteomic profiles among different regions of human carotid plaques in men and women

    PubMed Central

    Liang, Wenzhao; Ward, Liam J.; Karlsson, Helen; Ljunggren, Stefan A.; Li, Wei; Lindahl, Mats; Yuan, Xi-Ming

    2016-01-01

    The heterogeneity of atherosclerotic tissue has limited comprehension in proteomic and metabolomic analyses. To elucidate the functional implications, and differences between genders, of atherosclerotic lesion formation we investigated protein profiles from different regions of human carotid atherosclerotic arteries; internal control, fatty streak, plaque shoulder, plaque centre, and fibrous cap. Proteomic analysis was performed using 2-DE with MALDI-TOF, with validation using nLC-MS/MS. Protein mapping of 2-DE identified 52 unique proteins, including 15 previously unmapped proteins, of which 41 proteins were confirmed by nLC-MS/MS analysis. Expression levels of 18 proteins were significantly altered in plaque regions compared to the internal control region. Nine proteins showed site-specific alterations, irrespective of gender, with clear associations to extracellular matrix remodelling. Five proteins display gender-specific alterations with 2-DE, with two alterations validated by nLC-MS/MS. Gender differences in ferritin light chain and transthyretin were validated using both techniques. Validation of immunohistochemistry confirmed significantly higher levels of ferritin in plaques from male patients. Proteomic analysis of different plaque regions has reduced the effects of plaque heterogeneity, and significant differences in protein expression are determined in specific regions and between genders. These proteomes have functional implications in plaque progression and are of importance in understanding gender differences in atherosclerosis. PMID:27198765

  11. Proteomic profiling of lymphocytes in autoimmunity, inflammation and cancer

    PubMed Central

    2014-01-01

    Lymphocytes play important roles in the balance between body defense and noxious agents involved in a number of diseases, e.g. autoimmune diseases, allergic inflammation and cancer. The proteomic analyses have been applied to identify and validate disease-associated and disease-specific biomarkers for therapeutic strategies of diseases. The proteomic profiles of lymphocytes may provide more information to understand their functions and roles in the development of diseases, although proteomic approaches in lymphocytes are still limited. The present review overviewed the proteomics-based studies on lymphocytes to headlight the proteomic profiles of lymphocytes in diseases, such as autoimmune diseases, allergic inflammation and cancer, with a special focus on lung diseases. We will explore the potential significance of diagnostic biomarkers and therapeutic targets from the current status in proteomic studies of lymphocytes and discuss the value of the currently available proteomic methodologies in the lymphocytes research. PMID:24397796

  12. Breast tumor metastasis: analysis via proteomic profiling

    PubMed Central

    Goodison, Steve; Urquidi, Virginia

    2012-01-01

    The ability to predict the metastatic behavior of a patient’s cancer, as well as to detect and eradicate such recurrences, remain major clinical challenges in oncology. While many potential molecular biomarkers have been identified and tested previously, none have greatly improved the accuracy of specimen evaluation over routine histopathological criteria and, to date, they predict individual outcomes poorly. The ongoing development of high-throughput proteomic profiling technologies is opening new avenues for the investigation of cancer and, through application in tissue-based studies and animal models, will facilitate the identification of molecular signatures that are associated with breast tumor cell phenotype. The appropriate use of these approaches has the potential to provide efficient biomarkers, and to improve our knowledge of tumor biology. This, in turn, will enable the development of targeted therapeutics aimed at ameliorating the lethal dissemination of breast cancer. In this review, we focus on the accumulating proteomic signatures of breast tumor progression, particularly those that correlate with the occurrence of distant metastases, and discuss some of the expected future developments in the field. PMID:18532913

  13. Proteome profiling of keratinocytes transforming to malignancy.

    PubMed

    Paulitschke, Verena; Gerner, Christopher; Hofstätter, Elisabeth; Mohr, Thomas; Mayer, Rupert Laurenz; Pehamberger, Hubert; Kunstfeld, Rainer

    2015-02-01

    To shed light on the multistep process of squamous cell carcinoma development and the underlying pathologic mechanisms, we performed comparative proteome analysis of keratinocytes, keratinocytes stimulated with Il-1beta, and A431 epidermoid carcinoma cells. Fractionation of the cells into supernatant, nucleus, and cytoplasm was followed by protein separation, proteolytic digest, and nano-LC separation, and fragmentation using an ion trap mass spectrometer. Specific bioinformatics tools were used to generate a list of keratinocyte-specific proteins. Ninety percent of these proteins were found to be upregulated in keratinocytes versus the A431 cells. Classification of the identified proteins by biologic function and gene set enrichment analysis revealed that keratinocytes produced more proteins involved in cell differentiation, cell adhesion, cell junction, calcium ion, calmodulin binding, cytoskeleton organization, and cytokinesis, whereas A431 produced more proteins involved in cell cycle checkpoint, cell cycle process, RNA processing and transport, DNA damage and repair, RNA and DNA binding, and chromatin remodeling. The protein signatures of A431 and normal keratinocytes treated with IL-1beta showed marked similarity, confirming that inflammation is an important step in malignant transformation in nonmelanoma skin cancer. Thus, proteome profiling and bioinformatic processing may support the understanding of the underlying mechanisms, with the potential to facilitate development of early biomarkers and patient-tailored therapy. PMID:25395074

  14. Proteomic profile of edible bird's nest proteins.

    PubMed

    Liu, Xiaoqing; Lai, Xintian; Zhang, Shiwei; Huang, Xiuli; Lan, Quanxue; Li, Yun; Li, Bifang; Chen, Wei; Zhang, Qinlei; Hong, Dezhi; Yang, Guowu

    2012-12-26

    Edible bird's nest (EBN) is made of the swiftlets' saliva, which has attracted rather more attention owing to its nutritious and medical properties. Although protein constitutes the main composition and plays an important role in EBN, few studies have focused on the proteomic profile of EBN. The purpose of this study was to produce a proteomic map and clarify common EBN proteins. Liquid-phase isoelectric focusing (LIEF) was combined with two-dimensional electrophoresis (2-DE) for comprehensive analysis of EBN proteins. From 20 to 100 protein spots were detected on 2-DE maps of EBN samples from 15 different sources. The proteins were mainly distributed in four taxa (A, B, C, and D) according to their molecular mass. Taxa A and D both contained common proteins and proteins that may be considered another characteristic of EBN. Taxon A was identified using MALDI-TOF-TOF/MS and found to be homologous to acidic mammalian chitinase-like ( Meleagris gallopavo ), which is in glycosyl hydrolase family 18. PMID:23214475

  15. Proteomic global profiling for cancer biomarker discovery.

    PubMed

    Faca, Vitor; Wang, Hong; Hanash, Samir

    2009-01-01

    The ultimate goal of cancer molecular diagnostics is the development of simple tests to predict cancer risk, detect cancer early, classify tumors, and monitor response to therapy. Proteomics is well suited for these tasks. However, there are substantial challenges that need to be met to identify the most informative markers using proteomics. Approaches for in-depth quantitative proteomic analysis based on isotopic labeling and protein fractionation are presented in this chapter. PMID:19241042

  16. Proteomics Signature Profiling (PSP): A Novel Contextualization Approach for Cancer Proteomics

    PubMed Central

    2012-01-01

    Traditional proteomics analysis is plagued by the use of arbitrary thresholds resulting in large loss of information. We propose here a novel method in proteomics that utilizes all detected proteins. We demonstrate its efficacy in a proteomics screen of 5 and 7 liver cancer patients in the moderate and late stage, respectively. Utilizing biological complexes as a cluster vector, and augmenting it with submodules obtained from partitioning an integrated and cleaned protein–protein interaction network, we calculate a Proteomics Signature Profile (PSP) for each patient based on the hit rates of their reported proteins, in the absence of fold change thresholds, against the cluster vector. Using this, we demonstrated that moderate- and late-stage patients segregate with high confidence. We also discovered a moderate-stage patient who displayed a proteomics profile similar to other poor-stage patients. We identified significant clusters using a modified version of the SNet approach. Comparing our results against the Proteomics Expansion Pipeline (PEP) on which the same patient data was analyzed, we found good correlation. Building on this finding, we report significantly more clusters (176 clusters here compared to 70 in PEP), demonstrating the sensitivity of this approach. Gene Ontology (GO) terms analysis also reveals that the significant clusters are functionally congruent with the liver cancer phenotype. PSP is a powerful and sensitive method for analyzing proteomics profiles even when sample sizes are small. It does not rely on the ratio scores but, rather, whether a protein is detected or not. Although consistency of individual proteins between patients is low, we found the reported proteins tend to hit clusters in a meaningful and informative manner. By extracting this information in the form of a Proteomics Signature Profile, we confirm that this information is conserved and can be used for (1) clustering of patient samples, (2) identification of significant

  17. A novel profile biomarker diagnosis for mass spectral proteomics.

    PubMed

    Han, Henry

    2014-01-01

    Mass spectrometry based proteomics technologies have allowed for a great progress in identifying disease biomarkers for clinical diagnosis and prognosis. However, they face acute challenges from a data reproducibility standpoint, in that no two independent studies have been found to produce the same proteomic patterns. Such reproducibility issues cause the identified biomarker patterns to lose repeatability and prevent real clinical usage. In this work, we propose a profile biomarker approach to overcome this problem from a machine-learning viewpoint by developing a novel derivative component analysis (DCA). As an implicit feature selection algorithm, derivative component analysis enables the separation of true signals from red herrings by capturing subtle data behaviors and removing system noises from a proteomic profile. We further demonstrate its advantages in disease diagnosis by viewing input data as a profile biomarker. The results from our profile biomarker diagnosis suggest an effective solution to overcoming proteomics data's reproducibility problem, present an alternative method for biomarker discovery in proteomics, and provide a good candidate for clinical proteomic diagnosis. PMID:24297560

  18. Proteomics for Protein Expression Profiling in Neuroscience*

    PubMed Central

    Freeman, Willard M.; Hemby, Scott E.

    2013-01-01

    As the technology of proteomics moves from a theoretical approach to a practical reality, neuroscientists will have to determine the most appropriate applications for this technology. Neuroscientists will have to surmount difficulties particular to their research, such as limited sample amounts, heterogeneous cellular compositions in samples, and the fact that many proteins of interest are rare, hydrophobic proteins. This review examines protein isolation and protein fractionation and separation using two-dimensional electrophoresis (2-DE) and mass spectrometry proteomic methods. Methods for quantifying relative protein expression between samples (e.g., 2-DIGE, and ICAT) are also described. The coverage of the proteome, ability to detect membrane proteins, resource requirements, and quantitative reliability of different approaches is also discussed. Although there are many challenges in proteomic neuroscience, this field promises many rewards in the future. PMID:15176464

  19. Early life lead exposure causes gender-specific changes in the DNA methylation profile of DNA extracted from dried blood spots

    PubMed Central

    Sen, Arko; Heredia, Nicole; Senut, Marie-Claude; Hess, Matthew; Land, Susan; Qu, Wen; Hollacher, Kurt; Dereski, Mary O; Ruden, Douglas M

    2015-01-01

    Aims In this paper, we tested the hypothesis that early life lead (Pb) exposure associated DNA methylation (5mC) changes are dependent on the sex of the child and can serve as biomarkers for Pb exposure. Methods In this pilot study, we measured the 5mC profiles of DNA extracted from dried blood spots (DBS) in a cohort of 43 children (25 males and 18 females; ages from 3 months to 5 years) from Detroit. Result & Discussion We found that the effect of Pb-exposure on the 5-mC profiles can be separated into three subtypes: affected methylation loci which are conserved irrespective of the sex of the child (conserved); affected methylation loci unique to males (male-specific); and affected methylation loci unique to females (female-specific). PMID:26077427

  20. Derivative component analysis for mass spectral serum proteomic profiles

    PubMed Central

    2014-01-01

    Background As a promising way to transform medicine, mass spectrometry based proteomics technologies have seen a great progress in identifying disease biomarkers for clinical diagnosis and prognosis. However, there is a lack of effective feature selection methods that are able to capture essential data behaviors to achieve clinical level disease diagnosis. Moreover, it faces a challenge from data reproducibility, which means that no two independent studies have been found to produce same proteomic patterns. Such reproducibility issue causes the identified biomarker patterns to lose repeatability and prevents it from real clinical usage. Methods In this work, we propose a novel machine-learning algorithm: derivative component analysis (DCA) for high-dimensional mass spectral proteomic profiles. As an implicit feature selection algorithm, derivative component analysis examines input proteomics data in a multi-resolution approach by seeking its derivatives to capture latent data characteristics and conduct de-noising. We further demonstrate DCA's advantages in disease diagnosis by viewing input proteomics data as a profile biomarker via integrating it with support vector machines to tackle the reproducibility issue, besides comparing it with state-of-the-art peers. Results Our results show that high-dimensional proteomics data are actually linearly separable under proposed derivative component analysis (DCA). As a novel multi-resolution feature selection algorithm, DCA not only overcomes the weakness of the traditional methods in subtle data behavior discovery, but also suggests an effective resolution to overcoming proteomics data's reproducibility problem and provides new techniques and insights in translational bioinformatics and machine learning. The DCA-based profile biomarker diagnosis makes clinical level diagnostic performances reproducible across different proteomic data, which is more robust and systematic than the existing biomarker discovery based

  1. A gender-specific discriminator in Sprague-Dawley rat urine: the deployment of a metabolic profiling strategy for biomarker discovery and identification.

    PubMed

    Hodson, Mark P; Dear, Gordon J; Roberts, Andy D; Haylock, Claire L; Ball, Rachel J; Plumb, Robert S; Stumpf, Chris L; Griffin, Julian L; Haselden, John N

    2007-03-15

    The use of nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) as complementary analytical techniques for open metabolic profiling is illustrated in the context of defining urinary biochemical discriminators between male and female Sprague-Dawley rats. Subsequent to the discovery of a female-specific urinary discriminator by LC-MS, further LC, MS, and NMR methods have been applied in a coordinated effort to identify this urinary component. Thereafter, the biological relevance and context of the identified component, in this case a steroid metabolite, has been achieved. This approach will be deployed in future studies of disease, drug efficacy, and toxicity to discover and identify biologically relevant markers. PMID:17266915

  2. Plasma Proteome Profiling to Assess Human Health and Disease.

    PubMed

    Geyer, Philipp E; Kulak, Nils A; Pichler, Garwin; Holdt, Lesca M; Teupser, Daniel; Mann, Matthias

    2016-03-23

    Proteins in the circulatory system mirror an individual's physiology. In daily clinical practice, protein levels are generally determined using single-protein immunoassays. High-throughput, quantitative analysis using mass-spectrometry-based proteomics of blood, plasma, and serum would be advantageous but is challenging because of the high dynamic range of protein abundances. Here, we introduce a rapid and robust "plasma proteome profiling" pipeline. This single-run shotgun proteomic workflow does not require protein depletion and enables quantitative analysis of hundreds of plasma proteomes from 1 μl single finger pricks with 20 min gradients. The apolipoprotein family, inflammatory markers such as C-reactive protein, gender-related proteins, and >40 FDA-approved biomarkers are reproducibly quantified (CV <20% with label-free quantification). Furthermore, we functionally interpret a 1,000-protein, quantitative plasma proteome obtained by simple peptide pre-fractionation. Plasma proteome profiling delivers an informative portrait of a person's health state, and we envision its large-scale use in biomedicine. PMID:27135364

  3. Proteomic Profiling of Rat Thyroarytenoid Muscle

    ERIC Educational Resources Information Center

    Welham, Nathan V.; Marriott, Gerard; Bless, Diane M.

    2006-01-01

    Purpose: Proteomic methodologies offer promise in elucidating the systemwide cellular and molecular processes that characterize normal and diseased thyroarytenoid (TA) muscle. This study examined methodological issues central to the application of 2-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS-PAGE) to the study of…

  4. Proteomic profiling of Tectona grandis L. leaf.

    PubMed

    Quiala, Elisa; Cañal, María Jesús; Rodríguez, Roberto; Yagüe, Norma; Chávez, Maité; Barbón, Raúl; Valledor, Luis

    2012-04-01

    Tectona grandis L. (teak) is one of the premier hardwood timbers in the world, ranking at present in the top five tropical hardwood species in terms of worldwide plantation area. Characterization of the proteins present in teak leaves will provide a basis for the development of new tools aimed at assisting tree selection, the monitoring of plant propagation, and the certification of clonal and phenotypic identities. In this paper, we describe the extraction, separation, and identification of leaf proteins from T. grandis using a TCA/acetone protocol, 2DE, and MALDI-TOF. After TCA/acetone protein extraction of leaves, 998 well-resolved spots were detected in Coomassie-stained gels within the 10-114 kDa relative molecular mass (Mr) range at a pH ranging from 3 to 11. A total of 120 spots were digested and subjected to MS. Of these, 100 nonredundant protein species were successfully identified. Functional classification of the identified proteins revealed that proteins involved in photosynthesis, protein translation, and energy production were the most abundant. This work is the first high-throughput attempt to study the T. grandis leaf proteome and represents a stepping stone for further differential expression proteomic studies related to growth, development, biomass production, and culture-associated physiological responses. PMID:22522810

  5. Proteome profiling of Leishmania infantum promastigotes.

    PubMed

    Alcolea, Pedro J; Alonso, Ana; Larraga, Vicente

    2011-01-01

    A proteome analysis of the promastigote stage of the trypanosomatid parasite Leishmania infantum (MON-1 zymodeme) is described here for the first time. Total protein extracts were prepared at early logarithmic and stationary phases of replicate axenic cultures and processed by 2D electrophoresis (pH 3-10). A total of 28 differentially regulated proteins were identified by matrix-assisted laser desorption/ionization-tandem time of flight mass spectrometry. This approach has revealed that the electron transfer flavoprotein (ETF) and the eukaryotic elongation factor 1α (eEF1α) subunit have the same differential expression pattern at the protein and mRNA levels, up-regulation in the stationary phase. A low-molecular-weight isoform and an alternatively processed form of the eEF1α subunit have been detected. A 51 kDa subunit of replication factor A is up-regulated in dividing logarithmic promastigotes. None of the proteins described here shows opposite differential regulation values with the corresponding mRNA levels. Taken together with previous approaches to the proteome and the transcriptome, this report contributes to the elucidation of the differential regulation patterns of the ETF, the eEF1α subunit, the 40S ribosomal protein S12, α-tubulin and the T-complex protein 1 subunit γ throughout the life cycle of the parasites from the genus Leishmania. PMID:21569158

  6. Thermal proteome profiling monitors ligand interactions with cellular membrane proteins.

    PubMed

    Reinhard, Friedrich B M; Eberhard, Dirk; Werner, Thilo; Franken, Holger; Childs, Dorothee; Doce, Carola; Savitski, Maria Fälth; Huber, Wolfgang; Bantscheff, Marcus; Savitski, Mikhail M; Drewes, Gerard

    2015-12-01

    We extended thermal proteome profiling to detect transmembrane protein-small molecule interactions in cultured human cells. When we assessed the effects of detergents on ATP-binding profiles, we observed shifts in denaturation temperature for ATP-binding transmembrane proteins. We also observed cellular thermal shifts in pervanadate-induced T cell-receptor signaling, delineating the membrane target CD45 and components of the downstream pathway, and with drugs affecting the transmembrane transporters ATP1A1 and MDR1. PMID:26524241

  7. Quantitative proteomic profiling studies of pancreatic cancer stem cells.

    PubMed

    Dai, Lan; Li, Chen; Shedden, Kerby A; Lee, Cheong J; Li, Chenwei; Quoc, HuyVuong; Simeone, Diane M; Lubman, David M

    2010-07-01

    Analyzing subpopulations of tumor cells in tissue is a challenging subject in proteomic studies. Pancreatic cancer stem cells (CSCs) are such a group of cells that only constitute 0.2-0.8% of the total tumor cells but have been found to be the origin of pancreatic cancer carcinogenesis and metastasis. Global proteome profiling of pancreatic CSCs from xenograft tumors in mice is a promising way to unveil the molecular machinery underlying the signaling pathways. However, the extremely low availability of pancreatic tissue CSCs (around 10,000 cells per xenograft tumor or patient sample) has limited the utilization of currently standard proteomic approaches which do not work effectively with such a small amount of material. Herein, we describe the profiling of the proteome of pancreatic CSCs using a capillary scale shotgun technique by coupling offline capillary isoelectric focusing(cIEF) with nano reversed phase liquid chromatography(RPLC) followed by spectral counting peptide quantification. A whole cell lysate from 10,000 cells which corresponds to approximately 1 microg of protein material is equally divided for three repeated cIEF separations where around 300 ng of peptide material is used in each run. In comparison with a nontumorigenic tumor cell sample, among 1159 distinct proteins identified with FDR less than 0.2%, 169 differentially expressed proteins are identified after multiple testing corrections where 24% of the proteins are upregulated in the CSCs group. Ingenuity Pathway analysis of these differential expression signatures further suggests significant involvement of signaling pathways related to apoptosis, cell proliferation, inflammation, and metastasis. PMID:20486718

  8. Profiling the Proteome of Mycobacterium tuberculosis during Dormancy and Reactivation.

    PubMed

    Gopinath, Vipin; Raghunandanan, Sajith; Gomez, Roshna Lawrence; Jose, Leny; Surendran, Arun; Ramachandran, Ranjit; Pushparajan, Akhil Raj; Mundayoor, Sathish; Jaleel, Abdul; Kumar, Ramakrishnan Ajay

    2015-08-01

    Tuberculosis, caused by Mycobacterium tuberculosis, still remains a major global health problem. The main obstacle in eradicating this disease is the ability of this pathogen to remain dormant in macrophages, and then reactivate later under immuno-compromised conditions. The physiology of hypoxic nonreplicating M. tuberculosis is well-studied using many in vitro dormancy models. However, the physiological changes that take place during the shift from dormancy to aerobic growth (reactivation) have rarely been subjected to a detailed investigation. In this study, we developed an in vitro reactivation system by re-aerating the virulent laboratory strain of M. tuberculosis that was made dormant employing Wayne's dormancy model, and compared the proteome profiles of dormant and reactivated bacteria using label-free one-dimensional LC/MS/MS analysis. The proteome of dormant bacteria was analyzed at nonreplicating persistent stage 1 (NRP1) and stage 2 (NRP2), whereas that of reactivated bacteria was analyzed at 6 and 24 h post re-aeration. Proteome of normoxially grown bacteria served as the reference. In total, 1871 proteins comprising 47% of the M. tuberculosis proteome were identified, and many of them were observed to be expressed differentially or uniquely during dormancy and reactivation. The number of proteins detected at different stages of dormancy (764 at NRP1, 691 at NRP2) and reactivation (768 at R6 and 983 at R24) was very low compared with that of the control (1663). The number of unique proteins identified during normoxia, NRP1, NRP2, R6, and R24 were 597, 66, 56, 73, and 94, respectively. We analyzed various biological functions during these conditions. Fluctuation in the relative quantities of proteins involved in energy metabolism during dormancy and reactivation was the most significant observation we made in this study. Proteins that are up-regulated or uniquely expressed during reactivation from dormancy offer to be attractive targets for therapeutic

  9. Profiling the Proteome of Mycobacterium tuberculosis during Dormancy and Reactivation*

    PubMed Central

    Gopinath, Vipin; Raghunandanan, Sajith; Gomez, Roshna Lawrence; Jose, Leny; Surendran, Arun; Ramachandran, Ranjit; Pushparajan, Akhil Raj; Mundayoor, Sathish; Jaleel, Abdul; Kumar, Ramakrishnan Ajay

    2015-01-01

    Tuberculosis, caused by Mycobacterium tuberculosis, still remains a major global health problem. The main obstacle in eradicating this disease is the ability of this pathogen to remain dormant in macrophages, and then reactivate later under immuno-compromised conditions. The physiology of hypoxic nonreplicating M. tuberculosis is well-studied using many in vitro dormancy models. However, the physiological changes that take place during the shift from dormancy to aerobic growth (reactivation) have rarely been subjected to a detailed investigation. In this study, we developed an in vitro reactivation system by re-aerating the virulent laboratory strain of M. tuberculosis that was made dormant employing Wayne's dormancy model, and compared the proteome profiles of dormant and reactivated bacteria using label-free one-dimensional LC/MS/MS analysis. The proteome of dormant bacteria was analyzed at nonreplicating persistent stage 1 (NRP1) and stage 2 (NRP2), whereas that of reactivated bacteria was analyzed at 6 and 24 h post re-aeration. Proteome of normoxially grown bacteria served as the reference. In total, 1871 proteins comprising 47% of the M. tuberculosis proteome were identified, and many of them were observed to be expressed differentially or uniquely during dormancy and reactivation. The number of proteins detected at different stages of dormancy (764 at NRP1, 691 at NRP2) and reactivation (768 at R6 and 983 at R24) was very low compared with that of the control (1663). The number of unique proteins identified during normoxia, NRP1, NRP2, R6, and R24 were 597, 66, 56, 73, and 94, respectively. We analyzed various biological functions during these conditions. Fluctuation in the relative quantities of proteins involved in energy metabolism during dormancy and reactivation was the most significant observation we made in this study. Proteins that are up-regulated or uniquely expressed during reactivation from dormancy offer to be attractive targets for therapeutic

  10. Proteomic profiling change during the early development of silicosis disease

    PubMed Central

    Miao, Rongming; Ding, Bangmei; Zhang, Yingyi; Xia, Qian; Li, Yong

    2016-01-01

    Background Silicosis is one of several severe occupational diseases for which effective diagnostic tools during early development are currently unavailable. In this study we focused on proteomic profiling during the early stages of silicosis to investigate the pathophysiology and identify the proteins involved. Methods Two-dimensional (2D) gel electrophoresis and MALDI-TOF-MS were used to assess the proteomic differences between healthy individuals (HI), dust-exposed workers without silicosis (DEW) and silicosis patients (SP). Proteins abundances that differed by a factor of two-fold or greater were subjected to more detailed analysis, and enzyme linked to immunosorbent assay (ELISA) was employed to correlate with protein expression data. Results Compared with HI, 42 proteins were more abundant and 8 were less abundant in DEW, and these were also differentially accumulated in SP. Closer inspection revealed that serine protease granzyme A, alpha-1-B-glycoprotein (A1BG) and the T4 surface glycoprotein precursor (TSGP) were among the up-regulated proteins in DEW and SP. Significant changes in serine proteases, glycoproteins and proto-oncogenes may be associated with the response to cytotoxicity and infectious pathogens by activation of T cells, positive regulation of extracellular matrix structural constituents and immune response, and fibroblast proliferation. Up-regulation of cytokines included TNFs, interferon beta precursor, interleukin 6, atypical chemokine receptor 2, TNFR13BV, and mutant IL-17F may be involved in the increased and persistent immune response and fibrosis that occurred during silicosis development. Conclusions Granzymes, glycoproteins, cytokines and immune factors were dramatically involved in the immune response, metabolism, signal regulation and fibrosis during the early development of silicosis. Proteomic profiling has expanded our understanding of the pathogenesis of silicosis, and identified a number of targets that may be potential

  11. Comparative proteomic profiling of Hodgkin lymphoma cell lines.

    PubMed

    Vergara, D; Simeone, P; De Matteis, S; Carloni, S; Lanuti, P; Marchisio, M; Miscia, S; Rizzello, A; Napolitano, R; Agostinelli, C; Maffia, M

    2016-01-01

    Classical Hodgkin lymphoma (cHL) is a malignancy with complex pathogenesis. The hallmark of the disease is the presence of large mononucleated Hodgkin and bi- or multinucleated Reed/Sternberg (H/RS) cells. The origin of HRS cells in cHL is controversial as these cells show the coexpression of markers of several lineages. Using a proteomic approach, we compared the protein expression profile of cHL models of T- and B-cell derivation to find proteins differentially expressed in these cell lines. A total of 67 proteins were found differentially expressed between the two cell lines including metabolic proteins and proteins involved in the regulation of the cytoskeleton and/or cell migration, which were further validated by western blotting. Additionally, the expression of selected B- and T-cell antigens was also assessed by flow cytometry to reveal significant differences in the expression of different surface markers. Bioinformatics analysis was then applied to our dataset to find enriched pathways and networks, and to identify possible key regulators. In the present study, a proteomic approach was used to compare the protein expression profiles of two cHL cell lines. The identified proteins and/or networks, many of which not previously related to cHL, may be important to better define the pathogenesis of the disease, to identify novel diagnostic markers, and to design new therapeutic strategies. PMID:26588820

  12. Multivariate proteomic profiling identifies novel accessory proteins of coated vesicles

    PubMed Central

    Antrobus, Robin; Hirst, Jennifer; Bhumbra, Gary S.; Kozik, Patrycja; Jackson, Lauren P.; Sahlender, Daniela A.

    2012-01-01

    Despite recent advances in mass spectrometry, proteomic characterization of transport vesicles remains challenging. Here, we describe a multivariate proteomics approach to analyzing clathrin-coated vesicles (CCVs) from HeLa cells. siRNA knockdown of coat components and different fractionation protocols were used to obtain modified coated vesicle-enriched fractions, which were compared by stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative mass spectrometry. 10 datasets were combined through principal component analysis into a “profiling” cluster analysis. Overall, 136 CCV-associated proteins were predicted, including 36 new proteins. The method identified >93% of established CCV coat proteins and assigned >91% correctly to intracellular or endocytic CCVs. Furthermore, the profiling analysis extends to less well characterized types of coated vesicles, and we identify and characterize the first AP-4 accessory protein, which we have named tepsin. Finally, our data explain how sequestration of TACC3 in cytosolic clathrin cages causes the severe mitotic defects observed in auxilin-depleted cells. The profiling approach can be adapted to address related cell and systems biological questions. PMID:22472443

  13. Quantitative reactivity profiling predicts functional cysteines in proteomes

    PubMed Central

    Weerapana, Eranthie; Wang, Chu; Simon, Gabriel M.; Richter, Florian; Khare, Sagar; Dillon, Myles B.D.; Bachovchin, Daniel A.; Mowen, Kerri; Baker, David; Cravatt, Benjamin F.

    2010-01-01

    Cysteine is the most intrinsically nucleophilic amino acid in proteins, where its reactivity is tuned to perform diverse biochemical functions. The absence of a consensus sequence that defines functional cysteines in proteins has hindered their discovery and characterization. Here, we describe a proteomics method to quantitatively profile the intrinsic reactivity of cysteine residues en masse directly in native biological systems. Hyperreactivity was a rare feature among cysteines and found to specify a wide range of activities, including nucleophilic and reductive catalysis and sites of oxidative modification. Hyperreactive cysteines were identified in several proteins of uncharacterized function, including a residue conserved across eukaryotic phylogeny that we show is required for yeast viability and involved in iron-sulfur protein biogenesis. Finally, we demonstrate that quantitative reactivity profiling can also form the basis for screening and functional assignment of cysteines in computationally designed proteins, where it discriminated catalytically active from inactive cysteine hydrolase designs. PMID:21085121

  14. Quantitative reactivity profiling predicts functional cysteines in proteomes.

    PubMed

    Weerapana, Eranthie; Wang, Chu; Simon, Gabriel M; Richter, Florian; Khare, Sagar; Dillon, Myles B D; Bachovchin, Daniel A; Mowen, Kerri; Baker, David; Cravatt, Benjamin F

    2010-12-01

    Cysteine is the most intrinsically nucleophilic amino acid in proteins, where its reactivity is tuned to perform diverse biochemical functions. The absence of a consensus sequence that defines functional cysteines in proteins has hindered their discovery and characterization. Here we describe a proteomics method to profile quantitatively the intrinsic reactivity of cysteine residues en masse directly in native biological systems. Hyper-reactivity was a rare feature among cysteines and it was found to specify a wide range of activities, including nucleophilic and reductive catalysis and sites of oxidative modification. Hyper-reactive cysteines were identified in several proteins of uncharacterized function, including a residue conserved across eukaryotic phylogeny that we show is required for yeast viability and is involved in iron-sulphur protein biogenesis. We also demonstrate that quantitative reactivity profiling can form the basis for screening and functional assignment of cysteines in computationally designed proteins, where it discriminated catalytically active from inactive cysteine hydrolase designs. PMID:21085121

  15. Serum Proteomic Profiles In Subjects with Heavy Alcohol Abuse

    PubMed Central

    Liangpunsakul, Suthat; Lai, Xianyin; Ringham, Heather N.; Crabb, David W.; Witzmann, Frank A.

    2009-01-01

    Objectives The abuse of alcohol is a major public health problem, and the diagnosis and care of patients with alcohol abuse and dependence is hindered by the lack of tests that can detect dangerous levels of drinking or relapse during therapy. Gastroenterologists and other healthcare providers find it very challenging to obtain an accurate alcohol drinking history. We hypothesized that the effects of ethanol on numerous systems may well be reflected in changes in quantity or qualities of constituent or novel plasma proteins or protein fragments. Organ/tissue-specific proteins may be released into the blood stream when cells are injured by alcohol, or when systemic changes are induced by alcohol, and such proteins would be detected using a proteomic approach. The objective of this pilot study was to determine if there are plasma proteome profiles that correlate with heavy alcohol use. Methods Paired serum samples, before and after intensive alcohol treatment, were obtained from subjects who attended an outpatient alcohol treatment program. Serum proteomic profiles using MALDI –OTOF Mass Spectrometry were compared between pre- and post treatment samples. Results Of 16 subjects who enrolled in the study, 8 were females. The mean age of the study subjects was 49 yrs. The baseline laboratory data showed elevated AST (54 ± 37 IU/L), ALT (37 ± 19 IU/L), and MCV (99 ± 5 fl). Self-reported pre-treatment drinking levels for these subjects averaged 17 ± 7drinks/day and 103 ± 37 drinks/week. Mass spectrometry analyses showed a novel 5.9 kDa protein, a fragment of alpha fibrinogen, isoform 1, that might be might be a new novel marker for abusive alcohol drinking. Conclusions We have shown in this pilot study that several potential protein markers have appeared in mass spectral profiles and that they may be useful clinically to determine the status of alcohol drinking by MALDI –OTOF mass spectrometry, especially a fragment of alpha fibrinogen, isoform 1. However, a

  16. Proteomic profile response of Paracoccidioides lutzii to the antifungal argentilactone

    PubMed Central

    Prado, Renata S.; Bailão, Alexandre M.; Silva, Lívia C.; de Oliveira, Cecília M. A.; Marques, Monique F.; Silva, Luciano P.; Silveira-Lacerda, Elisângela P.; Lima, Aliny P.; Soares, Célia M.; Pereira, Maristela

    2015-01-01

    The dimorphic fungi Paracoccidioides spp. are the etiological agents of paracoccidioidomycosis (PCM), a mycosis of high incidence in Brazil. The toxicity of drug treatment and the emergence of resistant organisms have led to research for new candidates for drugs. In this study, we demonstrate that the natural product argentilactone was not cytotoxic or genotoxic to MRC5 cells at the IC50 concentration to the fungus. We also verified the proteomic profile of Paracoccidioides lutzii after incubation with argentilactone using a label free quantitative proteome nanoUPLC-MSE. The results of this study indicated that the fungus has a global metabolic adaptation in the presence of argentilactone. Enzymes of important pathways, such as glycolysis, the Krebs cycle and the glyoxylate cycle, were repressed, which drove the metabolism to the methylcytrate cycle and beta-oxidation. Proteins involved in cell rescue, defense and stress response were induced. In this study, alternative metabolic pathways adopted by the fungi were elucidated, helping to elucidate the course of action of the compound studied. PMID:26150808

  17. Quantitative proteomic profiling identifies protein correlates to EGFR kinase inhibition.

    PubMed

    Kani, Kian; Faca, Vitor M; Hughes, Lindsey D; Zhang, Wenxuan; Fang, Qiaojun; Shahbaba, Babak; Luethy, Roland; Erde, Jonathan; Schmidt, Joanna; Pitteri, Sharon J; Zhang, Qing; Katz, Jonathan E; Gross, Mitchell E; Plevritis, Sylvia K; McIntosh, Martin W; Jain, Anjali; Hanash, Samir; Agus, David B; Mallick, Parag

    2012-05-01

    Clinical oncology is hampered by lack of tools to accurately assess a patient's response to pathway-targeted therapies. Serum and tumor cell surface proteins whose abundance, or change in abundance in response to therapy, differentiates patients responding to a therapy from patients not responding to a therapy could be usefully incorporated into tools for monitoring response. Here, we posit and then verify that proteomic discovery in in vitro tissue culture models can identify proteins with concordant in vivo behavior and further, can be a valuable approach for identifying tumor-derived serum proteins. In this study, we use stable isotope labeling of amino acids in culture (SILAC) with proteomic technologies to quantitatively analyze the gefitinib-related protein changes in a model system for sensitivity to EGF receptor (EGFR)-targeted tyrosine kinase inhibitors. We identified 3,707 intracellular proteins, 1,276 cell surface proteins, and 879 shed proteins. More than 75% of the proteins identified had quantitative information, and a subset consisting of 400 proteins showed a statistically significant change in abundance following gefitinib treatment. We validated the change in expression profile in vitro and screened our panel of response markers in an in vivo isogenic resistant model and showed that these were markers of gefitinib response and not simply markers of phospho-EGFR downregulation. In doing so, we also were able to identify which proteins might be useful as markers for monitoring response and which proteins might be useful as markers for a priori prediction of response. PMID:22411897

  18. Quantitative Proteomic profiling identifies protein correlates to EGFR kinase inhibition

    PubMed Central

    Kani, Kian; Faca, Vitor M.; Hughes, Lindsey D.; Zhang, Wenxuan; Fang, Qiaojun; Shahbaba, Babak; Luethy, Roland; Erde, Jonathan; Schmidt, Joanna; Pitteri, Sharon J.; Zhang, Qing; Katz, Jonathan E.; Gross, Mitchell E.; Plevritis, Sylvia K.; McIntosh, Martin W.; Jain, Anjali; Hanash, Sam; Agus, David B.; Mallick, Parag

    2014-01-01

    Clinical oncology is hampered by a lack of tools to accurately assess a patient’s response to pathway-targeted therapies. Serum and tumor cell surface proteins whose abundance, or change in abundance in response to therapy, differentiates patients responding to a therapy from patients not-responding to a therapy could be usefully incorporated into tools for monitoring response. Here we posit and then verify that proteomic discovery in in vitro tissue culture models can identify proteins with concordant in vivo behavior and further, can be a valuable approach for identifying tumor-derived serum proteins. In this study we use Stable Isotope Labeling of Amino acids in Culture (SILAC) with proteomic technologies to quantitatively analyze the gefitinib-related protein changes in a model system for sensitivity to EGFR targeted tyrosine kinase inhibitors. We identified 3,707 intracellular proteins, 1,276 cell surface proteins, and 879 shed proteins. More than 75% of the proteins identified had quantitative information and a subset consisting of [400] proteins showed a statistically significant change in abundance following gefitinib treatment. We validated the change in expression profile in vitro and screened our panel of response markers in an in vivo isogenic resistant model and demonstrated that these were markers of gefitinib response and not simply markers of phospho-EGFR downregulation. In doing so, we also were able to identify which proteins might be useful as markers for monitoring response and which proteins might be useful as markers for a priori prediction of response. PMID:22411897

  19. Proteomic profile of mouse fibroblasts exposed to pure magnesium extract.

    PubMed

    Zhen, Zhen; Luthringer, Bérengère; Yang, Li; Xi, Tingfei; Zheng, Yufeng; Feyerabend, Frank; Willumeit, Regine; Lai, Chen; Ge, Zigang

    2016-12-01

    Magnesium and its alloys gain wide attention as degradable biomaterials. In order to reveal the molecular mechanism of the influence of biodegradable magnesium on cells, proteomics analysis was performed in this work. After mouse fibroblasts (L929) were cultured with or without Mg degradation products (Mg-extract) for 8, 24, and 48h, changes in protein expression profiles were obtained using isobaric tags for relative and absolute quantitation (iTRAQ) coupled two dimensional liquid chromatography-tandem mass spectrometry (2D LC MS/MS). A total of 867 proteins were identified (relying on at least two peptides). Compared to the control group, 205, 282, and 217 regulated proteins were identified at 8, 24, and 48h, respectively. 65 common proteins were up or down- regulated within all the three time points, which were involved in various physiological and metabolic activities. Consistent with viability, proliferation, and cell cycle analysis, stimulated energy metabolism as well as protein synthesis pathways were discussed, indicating a possible effect of Mg-extract on L929 proliferation. Furthermore, endocytosis and focal adhesion processes were also discussed. This proteomics study uncovers early cellular mechanisms triggered by Mg degradation products and highlights the cytocompatibility of biodegradable metallic materials for biomedical applications such as stents or orthopaedic implants. PMID:27612743

  20. HPLC-Chip/MS technology in proteomic profiling.

    PubMed

    Vollmer, Martin; van de Goor, Tom

    2009-01-01

    HPLC-chip/MS is a novel nanoflow analytical technology conducted on a microfabricated chip that allows for highly efficient HPLC separation and superior sensitive MS detection of complex proteomic mixtures. This is possible through on-chip preconcentration and separation with fluidic connection made automatically in a leak-tight fashion. Minimum precolumn and postcolumn peak dispersion and uncompromised ease of use result in compounds eluting in bands of only a few nanoliters. The chip is fabricated out of bio-inert polyimide-containing channels and integrated chip structures, such as an electrospray emitter, columns, and frits manufactured by laser ablation technology. Meanwhile, a variety of HPLC-chips differing in design and stationary phase are commercially available, which provide a comprehensive solution for applications in proteomics, glycomics, biomarker, and pharmaceutical discovery. The HPLC-chip can also be easily integrated into a multidimensional separation workflow where different orthogonal separation techniques are combined to solve a highly complex separation problems. In this chapter, we describe in detail the methodological chip usage and functionality and its application in the elucidation of the protein profile of human nucleoli. PMID:19488689

  1. PROTEOFORMER: deep proteome coverage through ribosome profiling and MS integration

    PubMed Central

    Crappé, Jeroen; Ndah, Elvis; Koch, Alexander; Steyaert, Sandra; Gawron, Daria; De Keulenaer, Sarah; De Meester, Ellen; De Meyer, Tim; Van Criekinge, Wim; Van Damme, Petra; Menschaert, Gerben

    2015-01-01

    An increasing amount of studies integrate mRNA sequencing data into MS-based proteomics to complement the translation product search space. However, several factors, including extensive regulation of mRNA translation and the need for three- or six-frame-translation, impede the use of mRNA-seq data for the construction of a protein sequence search database. With that in mind, we developed the PROTEOFORMER tool that automatically processes data of the recently developed ribosome profiling method (sequencing of ribosome-protected mRNA fragments), resulting in genome-wide visualization of ribosome occupancy. Our tool also includes a translation initiation site calling algorithm allowing the delineation of the open reading frames (ORFs) of all translation products. A complete protein synthesis-based sequence database can thus be compiled for mass spectrometry-based identification. This approach increases the overall protein identification rates with 3% and 11% (improved and new identifications) for human and mouse, respectively, and enables proteome-wide detection of 5′-extended proteoforms, upstream ORF translation and near-cognate translation start sites. The PROTEOFORMER tool is available as a stand-alone pipeline and has been implemented in the galaxy framework for ease of use. PMID:25510491

  2. Dataset of target mass spectromic proteome profiling for human chromosome 18.

    PubMed

    Ilgisonis, Ekaterina V; Kopylov, Arthur T; Zgoda, Victor G

    2016-09-01

    Proteome profiling is a type of quantitative analysis that reveals level of protein expression in the sample. Proteome profiling by using selected reaction monitoring is an approach for the Chromosome-centric Human Proteome Project (C-HPP). Here we describe dataset generated in the course of the pilot phase of Russian part of C-HPP, which was focused on human Chr 18 proteins. Proteome profiling was performed using stable isotope-labeled standards (SRM/SIS) for plasma, liver tissue and HepG2 cells. Dataset includes both positive and negative results of protein detection. These data were partly discussed in recent publications, "Chromosome 18 Transcriptome Profiling and Targeted Proteome Mapping in Depleted Plasma, Liver Tissue and HepG2 Cells" [1] and "Chromosome 18 transcriptoproteome of liver tissue and HepG2 Cells and targeted proteome mapping in depleted plasma: Update 2013" [2], supporting the accompanying publication "State of the Chromosome 18-centric HPP in 2016: Transcriptome and Proteome Profiling of Liver Tissue and HepG2 Cells" [3], and are deposited at the ProteomeXchange via the PASSEL repository with the dataset identifier PASSEL: PASS00697 for liver and HepG2 cell line. PMID:27595127

  3. A Chemical Proteomics Approach to Profiling the ATP-binding Proteome of Mycobacterium tuberculosis *

    PubMed Central

    Wolfe, Lisa M.; Veeraraghavan, Usha; Idicula-Thomas, Susan; Schürer, Stephan; Wennerberg, Krister; Reynolds, Robert; Besra, Gurdyal S.; Dobos, Karen M.

    2013-01-01

    Tuberculosis, caused by Mycobacterium tuberculosis, remains one of the leading causes of death worldwide despite extensive research, directly observed therapy using multidrug regimens, and the widespread use of a vaccine. The majority of patients harbor the bacterium in a state of metabolic dormancy. New drugs with novel modes of action are needed to target essential metabolic pathways in M. tuberculosis; ATP-competitive enzyme inhibitors are one such class. Previous screening efforts for ATP-competitive enzyme inhibitors identified several classes of lead compounds that demonstrated potent anti-mycobacterial efficacy as well as tolerable levels of toxicity in cell culture. In this report, a probe-based chemoproteomic approach was used to selectively profile the M. tuberculosis ATP-binding proteome in normally growing and hypoxic M. tuberculosis. From these studies, 122 ATP-binding proteins were identified in either metabolic state, and roughly 60% of these are reported to be essential for survival in vitro. These data are available through ProteomeXchange with identifier PXD000141. Protein families vital to the survival of the tubercle bacillus during hypoxia emerged from our studies. Specifically, along with members of the DosR regulon, several proteins involved in energy metabolism (Icl/Rv0468 and Mdh/Rv1240) and lipid biosynthesis (UmaA/Rv0469, DesA1/Rv0824c, and DesA2/Rv1094) were found to be differentially abundant in hypoxic versus normal growing cultures. These pathways represent a subset of proteins that may be relevant therapeutic targets for development of novel ATP-competitive antibiotics. PMID:23462205

  4. Quantitative proteomic profiling of human articular cartilage degradation in osteoarthritis.

    PubMed

    Lourido, Lucía; Calamia, Valentina; Mateos, Jesús; Fernández-Puente, Patricia; Fernández-Tajes, Juan; Blanco, Francisco J; Ruiz-Romero, Cristina

    2014-12-01

    Osteoarthritis (OA) is the most common rheumatic pathology and is characterized primarily by articular cartilage degradation. Despite its high prevalence, there is no effective therapy to slow disease progression or regenerate the damaged tissue. Therefore, new diagnostic and monitoring tests for OA are urgently needed, which would also promote the development of alternative therapeutic strategies. In the present study, we have performed an iTRAQ-based quantitative proteomic analysis of secretomes from healthy human articular cartilage explants, comparing their protein profile to those from unwounded (early disease) and wounded (advanced disease) zones of osteoarthritic tissue. This strategy allowed us to identify a panel of 76 proteins that are distinctively released by the diseased tissue. Clustering analysis allowed the classification of proteins according to their different profile of release from cartilage. Among these proteins, the altered release of osteoprotegerin (decreased in OA) and periostin (increased in OA), both involved in bone remodelling processes, was verified in further analyses. Moreover, periostin was also increased in the synovial fluid of OA patients. Altogether, the present work provides a novel insight into the mechanisms of human cartilage degradation and a number of new cartilage-characteristic proteins with possible biomarker value for early diagnosis and prognosis of OA. PMID:25383958

  5. Proteomic and Phospho-Proteomic Profile of Human Platelets in Basal, Resting State: Insights into Integrin Signaling

    PubMed Central

    Maiguel, Dony; Faridi, Mohd Hafeez; Barth, Constantinos J.; Salem, Saeed M.; Singhal, Mudita; Stoub, Darren; Krastins, Bryan; Ogihara, Mitsunori; Zaki, Mohammed J.; Gupta, Vineet

    2009-01-01

    During atherogenesis and vascular inflammation quiescent platelets are activated to increase the surface expression and ligand affinity of the integrin αIIbβ3 via inside-out signaling. Diverse signals such as thrombin, ADP and epinephrine transduce signals through their respective GPCRs to activate protein kinases that ultimately lead to the phosphorylation of the cytoplasmic tail of the integrin αIIbβ3 and augment its function. The signaling pathways that transmit signals from the GPCR to the cytosolic domain of the integrin are not well defined. In an effort to better understand these pathways, we employed a combination of proteomic profiling and computational analyses of isolated human platelets. We analyzed ten independent human samples and identified a total of 1507 unique proteins in platelets. This is the most comprehensive platelet proteome assembled to date and includes 190 membrane-associated and 262 phosphorylated proteins, which were identified via independent proteomic and phospho-proteomic profiling. We used this proteomic dataset to create a platelet protein-protein interaction (PPI) network and applied novel contextual information about the phosphorylation step to introduce limited directionality in the PPI graph. This newly developed contextual PPI network computationally recapitulated an integrin signaling pathway. Most importantly, our approach not only provided insights into the mechanism of integrin αIIbβ3 activation in resting platelets but also provides an improved model for analysis and discovery of PPI dynamics and signaling pathways in the future. PMID:19859549

  6. Advances and Challenges in Liquid Chromatography-Mass Spectrometry-Based Proteomics Profiling for Clinical Applications

    SciTech Connect

    Qian, Weijun; Jacobs, Jon M.; Liu, Tao; Camp, David G.; Smith, Richard D.

    2006-08-01

    The advances in proteomic technologies provide tremendous opportunities for applying these technologies in biomarker-related clinical applications; however, the unique characteristics of human biofluids such as high dynamic range in protein abundances and extreme complexity of human proteomes present tremendous challenges for current analytical technologies. In this review, we focus on summarizing the recent advances in LC-MS based proteomic profiling and its applications in clinical proteomics as well as the major challenges for implementing these technologies for more effective biomarker discovery. Over the last few years, tremendous efforts have been directed towards the development of more effective approaches for characterizing the human plasma/serum and other biofluid proteomes. The developments in immunodepletion and various fractionation approaches in combination with much improved LC-MS platforms have enabled the profiling of the plasma proteome with much greater dynamic range of coverage, allowing many proteins at low ng/mL levels being confidently identified. Despite the significant advances and efforts, the dynamic range of measurements or extent of proteome coverage, the confidence of peptide/protein identification, the accuracy of quantitation, the throughput of analysis, and the robustness of the present instrumentation are still among the major challenges for implementation of a proteomic profiling platform suitable for efficient clinical applications.

  7. Susceptibility to COPD: Differential Proteomic Profiling after Acute Smoking

    PubMed Central

    Franciosi, Lorenza; Postma, Dirkje S.; van den Berge, Maarten; Govorukhina, Natalia; Horvatovich, Peter L.; Fusetti, Fabrizia; Poolman, Bert; Lodewijk, Monique E.; Timens, Wim; Bischoff, Rainer; ten Hacken, Nick H. T.

    2014-01-01

    Cigarette smoking is the main risk factor for COPD (Chronic Obstructive Pulmonary Disease), yet only a subset of smokers develops COPD. Family members of patients with severe early-onset COPD have an increased risk to develop COPD and are therefore defined as “susceptible individuals”. Here we perform unbiased analyses of proteomic profiles to assess how “susceptible individuals” differ from age-matched “non-susceptible individuals” in response to cigarette smoking. Epithelial lining fluid (ELF) was collected at baseline and 24 hours after smoking 3 cigarettes in young individuals susceptible or non-susceptible to develop COPD and older subjects with established COPD. Controls at baseline were older healthy smoking and non-smoking individuals. Five samples per group were pooled and analysed by stable isotope labelling (iTRAQ) in duplicate. Six proteins were selected and validated by ELISA or immunohistochemistry. After smoking, 23 proteins increased or decreased in young susceptible individuals, 7 in young non-susceptible individuals, and 13 in COPD in the first experiment; 23 proteins increased or decreased in young susceptible individuals, 32 in young non-susceptible individuals, and 11 in COPD in the second experiment. SerpinB3 and Uteroglobin decreased after acute smoke exposure in young non-susceptible individuals exclusively, whereas Peroxiredoxin I, S100A9, S100A8, ALDH3A1 (Aldehyde dehydrogenase 3A1) decreased both in young susceptible and non-susceptible individuals, changes being significantly different between groups for Uteroglobin with iTRAQ and for Serpin B3 with iTRAQ and ELISA measures. Peroxiredoxin I, SerpinB3 and ALDH3A1 increased in COPD patients after smoking. We conclude that smoking induces a differential protein response in ELF of susceptible and non-susceptible young individuals, which differs from patients with established COPD. This is the first study applying unbiased proteomic profiling to unravel the underlying mechanisms

  8. Proteomic Profiling of a Biomimetic Drug Delivery Platform

    PubMed Central

    Corbo, Claudia; Parodi, Alessandro; Evangelopoulos, Michael; Engler, David A.; Matsunami, Risë K.; Engler, Anthony C.; Molinaro, Roberto; Scaria, Shilpa; Salvatore, Francesco; Tasciotti, Ennio

    2014-01-01

    Current delivery platforms are typically designed for prolonged circulation that favors superior accumulation of the payload in the targeted tissue. The design of efficient surface modifications determines both a longer circulation time and targeting abilities of particles. The optimization of synthesis protocols to efficiently combine targeting molecules and elements that allow for an increased circulation time can be challenging and almost impossible when several functional elements are needed. On the other hand, in the last decade, the development of bioinspired technologies was proposed as a new approach with which to increase particle safety, biocompatibility and targeting, while maintaining the synthesis protocols simple and reproducible. Recently, we developed a new drug delivery system inspired by the biology of immune cells called leukolike vector (LLV) and formed by a nanoporous silicon core and a shell derived from the leucocyte cell membrane. The goal of this study is to investigate the protein content of the LLV. Here we report the proteomic profiling of the LLV and demonstrate that our approach can be used to modify the surface of synthetic particles with more than 150 leukocyte membrane-associated proteins that determine particle safety, circulation time and targeting abilities towards inflamed endothelium. PMID:25382209

  9. Pathway analysis of kidney cancer using proteomics and metabolic profiling

    PubMed Central

    Perroud, Bertrand; Lee, Jinoo; Valkova, Nelly; Dhirapong, Amy; Lin, Pei-Yin; Fiehn, Oliver; Kültz, Dietmar; Weiss, Robert H

    2006-01-01

    Background Renal cell carcinoma (RCC) is the sixth leading cause of cancer death and is responsible for 11,000 deaths per year in the US. Approximately one-third of patients present with disease which is already metastatic and for which there is currently no adequate treatment, and no biofluid screening tests exist for RCC. In this study, we have undertaken a comprehensive proteomic analysis and subsequently a pathway and network approach to identify biological processes involved in clear cell RCC (ccRCC). We have used these data to investigate urinary markers of RCC which could be applied to high-risk patients, or to those being followed for recurrence, for early diagnosis and treatment, thereby substantially reducing mortality of this disease. Results Using 2-dimensional electrophoresis and mass spectrometric analysis, we identified 31 proteins which were differentially expressed with a high degree of significance in ccRCC as compared to adjacent non-malignant tissue, and we confirmed some of these by immunoblotting, immunohistochemistry, and comparison to published transcriptomic data. When evaluated by several pathway and biological process analysis programs, these proteins are demonstrated to be involved with a high degree of confidence (p values < 2.0 E-05) in glycolysis, propanoate metabolism, pyruvate metabolism, urea cycle and arginine/proline metabolism, as well as in the non-metabolic p53 and FAS pathways. In a pilot study using random urine samples from both ccRCC and control patients, we performed metabolic profiling and found that only sorbitol, a component of an alternative glycolysis pathway, is significantly elevated at 5.4-fold in RCC patients as compared to controls. Conclusion Extensive pathway and network analysis allowed for the discovery of highly significant pathways from a set of clear cell RCC samples. Knowledge of activation of these processes will lead to novel assays identifying their proteomic and/or metabolomic signatures in biofluids

  10. Platelets Proteomic Profiles of Acute Ischemic Stroke Patients

    PubMed Central

    Baykal, Ahmet Tarik; Sener, Azize

    2016-01-01

    Platelets play a crucial role in the pathogenesis of stroke and antiplatelet agents exist for its treatment and prevention. Through the use of LC-MS based protein expression profiling, platelets from stroke patients were analyzed and then correlated with the proteomic analyses results in the context of this disease. This study was based on patients who post ischemic stroke were admitted to hospital and had venous blood drawn within 24 hrs of the incidence. Label-free protein expression analyses of the platelets’ tryptic digest was performed in triplicate on a UPLC-ESI-qTOF-MS/MS system and ProteinLynx Global Server (v2.5, Waters) was used for tandem mass data extraction. The peptide sequences were searched against the reviewed homo sapiens database (www.uniprot.org) and the quantitation of protein variation was achieved through Progenesis LC-MS software (V4.0, Nonlinear Dynamics). These Label-free differential proteomics analysis of platelets ensured that 500 proteins were identified and 83 of these proteins were found to be statistically significant. The differentially expressed proteins are involved in various processes such as inflammatory response, cellular movement, immune cell trafficking, cell-to-cell signaling and interaction, hematological system development and function and nucleic acid metabolism. The expressions of myeloperoxidase, arachidonate 12-Lipoxygenase and histidine-rich glycoprotein are involved in cellular metabolic processes, crk-like protein and ras homolog gene family member A involved in cell signaling with vitronectin, thrombospondin 1, Integrin alpha 2b, and integrin beta 3 involved in cell adhesion. Apolipoprotein H, immunoglobulin heavy constant gamma 1 and immunoglobulin heavy constant gamma 3 are involved in structural, apolipoprotein A-I, and alpha-1-microglobulin/bikunin precursor is involved in transport, complement component 3 and clusterin is involved in immunity proteins as has been discussed. Our data provides an insight

  11. Platelets Proteomic Profiles of Acute Ischemic Stroke Patients.

    PubMed

    Cevik, Ozge; Baykal, Ahmet Tarik; Sener, Azize

    2016-01-01

    Platelets play a crucial role in the pathogenesis of stroke and antiplatelet agents exist for its treatment and prevention. Through the use of LC-MS based protein expression profiling, platelets from stroke patients were analyzed and then correlated with the proteomic analyses results in the context of this disease. This study was based on patients who post ischemic stroke were admitted to hospital and had venous blood drawn within 24 hrs of the incidence. Label-free protein expression analyses of the platelets' tryptic digest was performed in triplicate on a UPLC-ESI-qTOF-MS/MS system and ProteinLynx Global Server (v2.5, Waters) was used for tandem mass data extraction. The peptide sequences were searched against the reviewed homo sapiens database (www.uniprot.org) and the quantitation of protein variation was achieved through Progenesis LC-MS software (V4.0, Nonlinear Dynamics). These Label-free differential proteomics analysis of platelets ensured that 500 proteins were identified and 83 of these proteins were found to be statistically significant. The differentially expressed proteins are involved in various processes such as inflammatory response, cellular movement, immune cell trafficking, cell-to-cell signaling and interaction, hematological system development and function and nucleic acid metabolism. The expressions of myeloperoxidase, arachidonate 12-Lipoxygenase and histidine-rich glycoprotein are involved in cellular metabolic processes, crk-like protein and ras homolog gene family member A involved in cell signaling with vitronectin, thrombospondin 1, Integrin alpha 2b, and integrin beta 3 involved in cell adhesion. Apolipoprotein H, immunoglobulin heavy constant gamma 1 and immunoglobulin heavy constant gamma 3 are involved in structural, apolipoprotein A-I, and alpha-1-microglobulin/bikunin precursor is involved in transport, complement component 3 and clusterin is involved in immunity proteins as has been discussed. Our data provides an insight into

  12. The Urine Proteome Profile Is Different in Neuromyelitis Optica Compared to Multiple Sclerosis: A Clinical Proteome Study

    PubMed Central

    Kristensen, Lars P.; Burton, Mark; Csepany, Tunde; Simo, Magdolna; Dioszeghy, Peter; Sejbaek, Tobias; Grebing, Manuela; Heegaard, Niels H. H.; Illes, Zsolt

    2015-01-01

    Objectives Inflammatory demyelinating diseases of the CNS comprise a broad spectrum of diseases like neuromyelitis optica (NMO), NMO spectrum disorders (NMO-SD) and multiple sclerosis (MS). Despite clear classification criteria, differentiation can be difficult. We hypothesized that the urine proteome may differentiate NMO from MS. Methods The proteins in urine samples from anti-aquaporin 4 (AQP4) seropositive NMO/NMO-SD patients (n = 32), patients with MS (n = 46) and healthy subjects (HS, n = 31) were examined by quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) after trypsin digestion and iTRAQ labelling. Immunoglobulins (Ig) in the urine were validated by nephelometry in an independent cohort (n = 9–10 pr. groups). Results The analysis identified a total of 1112 different proteins of which 333 were shared by all 109 subjects. Cluster analysis revealed differences in the urine proteome of NMO/NMO-SD compared to HS and MS. Principal component analysis also suggested that the NMO/NMO-SD proteome profile was useful for classification. Multivariate regression analysis revealed a 3-protein profile for the NMO/NMO-SD versus HS discrimination, a 6-protein profile for NMO/NMO-SD versus MS discrimination and an 11-protein profile for MS versus HS discrimination. All protein panels yielded highly significant ROC curves (AUC in all cases >0.85, p≤0.0002). Nephelometry confirmed the presence of increased Ig-light chains in the urine of patients with NMO/NMO-SD. Conclusion The urine proteome profile of patients with NMO/NMO-SD is different from MS and HS. This may reflect differences in the pathogenesis of NMO/NMO-SD versus MS and suggests that urine may be a potential source of biomarkers differentiating NMO/NMO-SD from MS. PMID:26460890

  13. Functional Classification of Cellular Proteome Profiles Support the Identification of Drug Resistance Signatures in Melanoma Cells

    PubMed Central

    2013-01-01

    Drug resistance is a major obstacle in melanoma treatment. Recognition of specific resistance patterns, the understanding of the patho-physiology of drug resistance, and identification of remaining options for individual melanoma treatment would greatly improve therapeutic success. We performed mass spectrometry-based proteome profiling of A375 melanoma cells and HeLa cells characterized as sensitive to cisplatin in comparison to cisplatin resistant M24met and TMFI melanoma cells. Cells were fractionated into cytoplasm, nuclei and secretome and the proteome profiles classified according to Gene Ontology. The cisplatin resistant cells displayed increased expression of lysosomal as well as Ca2+ ion binding and cell adherence proteins. These findings were confirmed using Lysotracker Red staining and cell adhesion assays with a panel of extracellular matrix proteins. To discriminate specific survival proteins, we selected constitutively expressed proteins of resistant M24met cells which were found expressed upon challenging the sensitive A375 cells. Using the CPL/MUW proteome database, the selected lysosomal, cell adherence and survival proteins apparently specifying resistant cells were narrowed down to 47 proteins representing a potential resistance signature. These were tested against our proteomics database comprising more than 200 different cell types/cell states for its predictive power. We provide evidence that this signature enables the automated assignment of resistance features as readout from proteome profiles of any human cell type. Proteome profiling and bioinformatic processing may thus support the understanding of drug resistance mechanism, eventually guiding patient tailored therapy. PMID:23713901

  14. Proteomics and the Analysis of Proteomic Data: 2013 Overview of Current Protein-Profiling Technologies

    PubMed Central

    Bruce, Can; Stone, Kathryn; Gulcicek, Erol; Williams, Kenneth

    2013-01-01

    Mass spectrometry has become a major tool in the study of proteomes. The analysis of proteolytic peptides and their fragment ions by this technique enables the identification and quantitation of the precursor proteins in a mixture. However, deducing chemical structures and then protein sequences from mass-to-charge ratios is a challenging computational task. Software tools incorporating powerful algorithms and statistical methods improved our ability to process the large quantities of proteomics data. Repositories of spectral data make both data analysis and experimental design more efficient. New approaches in quantitative and statistical proteomics make possible a greater coverage of the proteome, the identification of more post-translational modifications and a greater sensitivity in the quantitation of targeted proteins. PMID:23504934

  15. Proteomic profiling of Beta vulgaris leaves during rhizomania compatible interactions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rhizomania severely impacts sugarbeet (Beta vulgaris) production throughout the world, and is widely prevalent in most sugarbeet growing regions. Initial efforts to characterize proteome changes focused primarily on identifying putative host factors that elicit resistant interactions with Beet Necr...

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

    PubMed

    Matallana-Surget, Sabine; Derock, Jérémy; Leroy, Baptiste; Badri, Hanène; Deschoenmaeker, Frédéric; Wattiez, Ruddy

    2014-01-01

    The filamentous cyanobacterium Arthrospira platensis has a long history of use as a food supply and it has been used by the European Space Agency in the MELiSSA project, an artificial microecosystem which supports life during long-term manned space missions. This study assesses progress in the field of cyanobacterial shotgun proteomics and light/dark diurnal cycles by focusing on Arthrospira platensis. Several fractionation workflows including gel-free and gel-based protein/peptide fractionation procedures were used and combined with LC-MS/MS analysis, enabling the overall identification of 1306 proteins, which represents 21% coverage of the theoretical proteome. A total of 30 proteins were found to be significantly differentially regulated under light/dark growth transition. Interestingly, most of the proteins showing differential abundance were related to photosynthesis, the Calvin cycle and translation processes. A novel aspect and major achievement of this work is the successful improvement of the cyanobacterial proteome coverage using a 3D LC-MS/MS approach, based on an immobilized metal affinity chromatography, a suitable tool that enabled us to eliminate the most abundant protein, the allophycocyanin. We also demonstrated that cell growth follows a light/dark cycle in A. platensis. This preliminary proteomic study has highlighted new characteristics of the Arthrospira platensis proteome in terms of diurnal regulation. PMID:24914774

  17. Proteome-Wide Analysis and Diel Proteomic Profiling of the Cyanobacterium Arthrospira platensis PCC 8005

    PubMed Central

    Matallana-Surget, Sabine; Derock, Jérémy; Leroy, Baptiste; Badri, Hanène; Deschoenmaeker, Frédéric; Wattiez, Ruddy

    2014-01-01

    The filamentous cyanobacterium Arthrospira platensis has a long history of use as a food supply and it has been used by the European Space Agency in the MELiSSA project, an artificial microecosystem which supports life during long-term manned space missions. This study assesses progress in the field of cyanobacterial shotgun proteomics and light/dark diurnal cycles by focusing on Arthrospira platensis. Several fractionation workflows including gel-free and gel-based protein/peptide fractionation procedures were used and combined with LC-MS/MS analysis, enabling the overall identification of 1306 proteins, which represents 21% coverage of the theoretical proteome. A total of 30 proteins were found to be significantly differentially regulated under light/dark growth transition. Interestingly, most of the proteins showing differential abundance were related to photosynthesis, the Calvin cycle and translation processes. A novel aspect and major achievement of this work is the successful improvement of the cyanobacterial proteome coverage using a 3D LC-MS/MS approach, based on an immobilized metal affinity chromatography, a suitable tool that enabled us to eliminate the most abundant protein, the allophycocyanin. We also demonstrated that cell growth follows a light/dark cycle in A. platensis. This preliminary proteomic study has highlighted new characteristics of the Arthrospira platensis proteome in terms of diurnal regulation. PMID:24914774

  18. Proteomic profiling reveals insights into Triticeae stigma development and function.

    PubMed

    Nazemof, Nazila; Couroux, Philippe; Rampitsch, Christof; Xing, Tim; Robert, Laurian S

    2014-11-01

    To our knowledge, this study represents the first high-throughput characterization of a stigma proteome in the Triticeae. A total of 2184 triticale mature stigma proteins were identified using three different gel-based approaches combined with mass spectrometry. The great majority of these proteins are described in a Triticeae stigma for the first time. These results revealed many proteins likely to play important roles in stigma development and pollen-stigma interactions, as well as protection against biotic and abiotic stresses. Quantitative comparison of the triticale stigma transcriptome and proteome showed poor correlation, highlighting the importance of having both types of analysis. This work makes a significant contribution towards the elucidation of the Triticeae stigma proteome and provides novel insights into its role in stigma development and function. PMID:25170101

  19. Proteomic profiling reveals insights into Triticeae stigma development and function

    PubMed Central

    Nazemof, Nazila; Couroux, Philippe; Rampitsch, Christof; Xing, Tim; Robert, Laurian S.

    2014-01-01

    To our knowledge, this study represents the first high-throughput characterization of a stigma proteome in the Triticeae. A total of 2184 triticale mature stigma proteins were identified using three different gel-based approaches combined with mass spectrometry. The great majority of these proteins are described in a Triticeae stigma for the first time. These results revealed many proteins likely to play important roles in stigma development and pollen–stigma interactions, as well as protection against biotic and abiotic stresses. Quantitative comparison of the triticale stigma transcriptome and proteome showed poor correlation, highlighting the importance of having both types of analysis. This work makes a significant contribution towards the elucidation of the Triticeae stigma proteome and provides novel insights into its role in stigma development and function. PMID:25170101

  20. Comparative Bioinformatics Analyses and Profiling of Lysosome-Related Organelle Proteomes

    PubMed Central

    Hu, Zhang-Zhi; Valencia, Julio C.; Huang, Hongzhan; Chi, An; Shabanowitz, Jeffrey; Hearing, Vincent J.; Appella, Ettore; Wu, Cathy

    2007-01-01

    Complete and accurate profiling of cellular organelle proteomes, while challenging, is important for the understanding of detailed cellular processes at the organelle level. Mass spectrometry technologies coupled with bioinformatics analysis provide an effective approach for protein identification and functional interpretation of organelle proteomes. In this study, we have compiled human organelle reference datasets from large-scale proteomic studies and protein databases for 7 lysosome-related organelles (LROs), as well as the endoplasmic reticulum and mitochondria, for comparative organelle proteome analysis. Heterogeneous sources of human organelle proteins and rodent homologs are mapped to human UniProtKB protein entries based on ID and/or peptide mappings, followed by functional annotation and categorization using the iProXpress proteomic expression analysis system. Cataloging organelle proteomes allows close examination of both shared and unique proteins among various LROs and reveals their functional relevance. The proteomic comparisons show that LROs are a closely related family of organelles. The shared proteins indicate the dynamic and hybrid nature of LROs, while the unique transmembrane proteins may represent additional candidate marker proteins for LROs. This comparative analysis, therefore, provides a basis for hypothesis formulation and experimental validation of organelle proteins and their functional roles. PMID:17375895

  1. Comparative bioinformatics analyses and profiling of lysosome-related organelle proteomes

    NASA Astrophysics Data System (ADS)

    Hu, Zhang-Zhi; Valencia, Julio C.; Huang, Hongzhan; Chi, An; Shabanowitz, Jeffrey; Hearing, Vincent J.; Appella, Ettore; Wu, Cathy

    2007-01-01

    Complete and accurate profiling of cellular organelle proteomes, while challenging, is important for the understanding of detailed cellular processes at the organelle level. Mass spectrometry technologies coupled with bioinformatics analysis provide an effective approach for protein identification and functional interpretation of organelle proteomes. In this study, we have compiled human organelle reference datasets from large-scale proteomic studies and protein databases for seven lysosome-related organelles (LROs), as well as the endoplasmic reticulum and mitochondria, for comparative organelle proteome analysis. Heterogeneous sources of human organelle proteins and rodent homologs are mapped to human UniProtKB protein entries based on ID and/or peptide mappings, followed by functional annotation and categorization using the iProXpress proteomic expression analysis system. Cataloging organelle proteomes allows close examination of both shared and unique proteins among various LROs and reveals their functional relevance. The proteomic comparisons show that LROs are a closely related family of organelles. The shared proteins indicate the dynamic and hybrid nature of LROs, while the unique transmembrane proteins may represent additional candidate marker proteins for LROs. This comparative analysis, therefore, provides a basis for hypothesis formulation and experimental validation of organelle proteins and their functional roles.

  2. Proteome-Wide Profiling of Targets of Cysteine reactive Small Molecules by Using Ethynyl Benziodoxolone Reagents.

    PubMed

    Abegg, Daniel; Frei, Reto; Cerato, Luca; Prasad Hari, Durga; Wang, Chao; Waser, Jerome; Adibekian, Alexander

    2015-09-01

    In this study, we present a highly efficient method for proteomic profiling of cysteine residues in complex proteomes and in living cells. Our method is based on alkynylation of cysteines in complex proteomes using a "clickable" alkynyl benziodoxolone bearing an azide group. This reaction proceeds fast, under mild physiological conditions, and with a very high degree of chemoselectivity. The formed azide-capped alkynyl-cysteine adducts are readily detectable by LC-MS/MS, and can be further functionalized with TAMRA or biotin alkyne via CuAAC. We demonstrate the utility of alkynyl benziodoxolones for chemical proteomics applications by identifying the proteomic targets of curcumin, a diarylheptanoid natural product that was and still is part of multiple human clinical trials as anticancer agent. Our results demonstrate that curcumin covalently modifies several key players of cellular signaling and metabolism, most notably the enzyme casein kinase I gamma. We anticipate that this new method for cysteine profiling will find broad application in chemical proteomics and drug discovery. PMID:26211368

  3. Chemical proteomic probes for profiling cytochrome P450 activities and drug interactions in vivo

    PubMed Central

    Wright, Aaron T.; Cravatt, Benjamin F.

    2007-01-01

    The cytochrome P450 (P450) superfamily metabolizes many endogenous signaling molecules and drugs. P450 enzymes are regulated by post-translational mechanisms in vivo, which hinders their functional characterization by conventional genomic or proteomic methods. Here, we describe a chemical proteomic strategy to profile P450 activities directly in living systems. Derivatization of a mechanism-based inhibitor with a “clickable” handle provided an activity-based probe that labels multiple P450s both in proteomic extracts and in vivo. This probe was used to record alterations in liver P450 activities triggered by chemical agents, including inducers of P450 expression and direct P450 inhibitors. The chemical proteomic strategy described herein thus offers a versatile method to monitor P450 activities and small molecule interactions in any biological system and, through doing so, should facilitate the functional characterization of this large and diverse enzyme class. PMID:17884636

  4. The proteome of Hypobaric Induced Hypoxic Lung: Insights from Temporal Proteomic Profiling for Biomarker Discovery

    PubMed Central

    Ahmad, Yasmin; Sharma, Narendra K.; Ahmad, Mohammad Faiz; Sharma, Manish; Garg, Iti; Srivastava, Mousami; Bhargava, Kalpana

    2015-01-01

    Exposure to high altitude induces physiological responses due to hypoxia. Lungs being at the first level to face the alterations in oxygen levels are critical to counter and balance these changes. Studies have been done analysing pulmonary proteome alterations in response to exposure to hypobaric hypoxia. However, such studies have reported the alterations at specific time points and do not reflect the gradual proteomic changes. These studies also identify the various biochemical pathways and responses induced after immediate exposure and the resolution of these effects in challenge to hypobaric hypoxia. In the present study, using 2-DE/MS approach, we attempt to resolve these shortcomings by analysing the proteome alterations in lungs in response to different durations of exposure to hypobaric hypoxia. Our study thus highlights the gradual and dynamic changes in pulmonary proteome following hypobaric hypoxia. For the first time, we also report the possible consideration of SULT1A1, as a biomarker for the diagnosis of high altitude pulmonary edema (HAPE). Higher SULT1A1 levels were observed in rats as well as in humans exposed to high altitude, when compared to sea-level controls. This study can thus form the basis for identifying biomarkers for diagnostic and prognostic purposes in responses to hypobaric hypoxia. PMID:26022216

  5. Proteomic Profiling of Bladders from Mice Exposed with Sodium Arsenite

    EPA Science Inventory

    Arsenic, an environmental contaminant, has been linked with cancer of the bladder in humans. To study the mode of action of arsenic, female CH3 mice were exposed to 85 ppm sodium arsenite in their drinking water for 30 days. Following the exposure a comparative proteomic analysis...

  6. Proteome Profile and Quantitative Proteomic Analysis of Buffalo (Bubalusbubalis) Follicular Fluid during Follicle Development.

    PubMed

    Fu, Qiang; Huang, Yulin; Wang, Zhiqiang; Chen, Fumei; Huang, Delun; Lu, Yangqing; Liang, Xianwei; Zhang, Ming

    2016-01-01

    Follicular fluid (FF) accumulates in the antrum of the ovarian follicle and provides the microenvironment for oocyte development. FF plays an important role in follicle growth and oocyte maturation. The FF provides a unique window to investigate the processes occurring during buffalo follicular development. The observed low quality of buffalo oocytes may arise from the poor follicular microenvironment. Investigating proteins found in buffalo FF (BFF) should provide insight into follicular development processes and provide further understanding of intra-follicular maturation and oocytes quality. Here, a proteomic-based approach was used to analyze the proteome of BFF. SDS-PAGE separation combined with mass spectrometry was used to generate the proteomic dataset. In total, 363 proteins were identified and classified by Gene Ontology terms. The proteins were assigned to 153 pathways, including signaling pathways. To evaluate difference in proteins expressed between BFF with different follicle size (small, <4 mm; and large, >8 mm), a quantitative proteomic analysis based on multi-dimensional liquid chromatography pre-fractionation tandem Orbitrap mass spectrometry identification was performed. Eleven differentially expressed proteins (six downregulated and five upregulated in large BFF) were identified and assigned to a variety of functional processes, including serine protease inhibition, oxidation protection and the complement cascade system. Three differentially expressed proteins, Vimentin, Peroxiredoxin-1 and SERPIND1, were verified by Western blotting, consistent with the quantitative proteomics results. Our datasets offers new information about proteins present in BFF and should facilitate the development of new biomarkers. These differentially expressed proteins illuminate the size-dependent protein changes in follicle microenvironment. PMID:27136540

  7. Proteome Profile and Quantitative Proteomic Analysis of Buffalo (Bubalusbubalis) Follicular Fluid during Follicle Development

    PubMed Central

    Fu, Qiang; Huang, Yulin; Wang, Zhiqiang; Chen, Fumei; Huang, Delun; Lu, Yangqing; Liang, Xianwei; Zhang, Ming

    2016-01-01

    Follicular fluid (FF) accumulates in the antrum of the ovarian follicle and provides the microenvironment for oocyte development. FF plays an important role in follicle growth and oocyte maturation. The FF provides a unique window to investigate the processes occurring during buffalo follicular development. The observed low quality of buffalo oocytes may arise from the poor follicular microenvironment. Investigating proteins found in buffalo FF (BFF) should provide insight into follicular development processes and provide further understanding of intra-follicular maturation and oocytes quality. Here, a proteomic-based approach was used to analyze the proteome of BFF. SDS-PAGE separation combined with mass spectrometry was used to generate the proteomic dataset. In total, 363 proteins were identified and classified by Gene Ontology terms. The proteins were assigned to 153 pathways, including signaling pathways. To evaluate difference in proteins expressed between BFF with different follicle size (small, <4 mm; and large, >8 mm), a quantitative proteomic analysis based on multi-dimensional liquid chromatography pre-fractionation tandem Orbitrap mass spectrometry identification was performed. Eleven differentially expressed proteins (six downregulated and five upregulated in large BFF) were identified and assigned to a variety of functional processes, including serine protease inhibition, oxidation protection and the complement cascade system. Three differentially expressed proteins, Vimentin, Peroxiredoxin-1 and SERPIND1, were verified by Western blotting, consistent with the quantitative proteomics results. Our datasets offers new information about proteins present in BFF and should facilitate the development of new biomarkers. These differentially expressed proteins illuminate the size-dependent protein changes in follicle microenvironment. PMID:27136540

  8. Dietary ractopamine influences sarcoplasmic proteome profile of pork Longissimus thoracis.

    PubMed

    Costa-Lima, Bruno R C; Suman, Surendranath P; Li, Shuting; Beach, Carol M; Silva, Teofilo J P; Silveira, Expedito T F; Bohrer, Benjamin M; Boler, Dustin D

    2015-05-01

    Dietary ractopamine improves pork leanness, whereas its effect on sarcoplasmic proteome has not been characterized. Therefore, the influence of ractopamine on sarcoplasmic proteome of post-mortem pork Longissimus thoracis muscle was examined. Longissimus thoracis samples were collected from carcasses (24 h post-mortem) of purebred Berkshire barrows (n=9) managed in mixed-sex pens and fed finishing diets containing ractopamine (RAC; 7.4 mg/kg for 14 days followed by 10.0 mg/kg for 14 days) or without ractopamine for 28 days (CON). Sarcoplasmic proteome was analyzed using two-dimensional electrophoresis and mass spectrometry. Nine protein spots were differentially abundant between RAC and CON groups. Glyceraldehyde-3-phosphate dehydrogenase and phosphoglucomutase-1 were over-abundant in CON, whereas serum albumin, carbonic anhydrase 3, L-lactate dehydrogenase A chain, fructose-bisphosphate aldolase A, and myosin light chain 1/3 were over-abundant in RAC. These results suggest that ractopamine influences the abundance of enzymes involved in glycolytic metabolism, and the differential abundance of glycolytic enzymes could potentially influence the conversion of muscle to meat. PMID:25576742

  9. Mass Spectrometry–based Proteomic Profiling of Lung Cancer

    PubMed Central

    Ocak, Sebahat; Chaurand, Pierre; Massion, Pierre P.

    2009-01-01

    In an effort to further our understanding of lung cancer biology and to identify new candidate biomarkers to be used in the management of lung cancer, we need to probe these tissues and biological fluids with tools that address the biology of lung cancer directly at the protein level. Proteins are responsible of the function and phenotype of cells. Cancer cells express proteins that distinguish them from normal cells. Proteomics is defined as the study of the proteome, the complete set of proteins produced by a species, using the technologies of large-scale protein separation and identification. As a result, new technologies are being developed to allow the rapid and systematic analysis of thousands of proteins. The analytical advantages of mass spectrometry (MS), including sensitivity and high-throughput, promise to make it a mainstay of novel biomarker discovery to differentiate cancer from normal cells and to predict individuals likely to develop or recur with lung cancer. In this review, we summarize the progress made in clinical proteomics as it applies to the management of lung cancer. We will focus our discussion on how MS approaches may advance the areas of early detection, response to therapy, and prognostic evaluation. PMID:19349484

  10. Simple and Integrated Spintip-Based Technology Applied for Deep Proteome Profiling.

    PubMed

    Chen, Wendong; Wang, Shuai; Adhikari, Subash; Deng, Zuhui; Wang, Lingjue; Chen, Lan; Ke, Mi; Yang, Pengyuan; Tian, Ruijun

    2016-05-01

    Great efforts have been taken for developing high-sensitive mass spectrometry (MS)-based proteomic technologies, among which sample preparation is one of the major focus. Here, a simple and integrated spintip-based proteomics technology (SISPROT) consisting of strong cation exchange beads and C18 disk in one pipet tip was developed. Both proteomics sample preparation steps, including protein preconcentration, reduction, alkylation, and digestion, and reversed phase (RP)-based desalting and high-pH RP-based peptide fractionation can be achieved in a fully integrated manner for the first time. This easy-to-use technology achieved high sensitivity with negligible sample loss. Proteomic analysis of 2000 HEK 293 cells readily identified 1270 proteins within 1.4 h of MS time, while 7826 proteins were identified when 100000 cells were processed and analyzed within only 22 h of MS time. More importantly, the SISPROT can be easily multiplexed on a standard centrifuge with good reproducibility (Pearson correlation coefficient > 0.98) for both single-shot analysis and deep proteome profiling with five-step high-pH RP fractionation. The SISPROT was exemplified by the triplicate analysis of 100000 stem cells from human exfoliated deciduous teeth (SHED). This led to the identification of 9078 proteins containing 3771 annotated membrane proteins, which was the largest proteome data set for dental stem cells reported to date. We expect that the SISPROT will be well suited for deep proteome profiling for fewer than 100000 cells and applied for translational studies where multiplexed technology with good label-free quantification precision is required. PMID:27062885

  11. Investigating the Correspondence Between Transcriptomic and Proteomic Expression Profiles Using Coupled Cluster Models.

    SciTech Connect

    Rogers, Simon; Girolami, Mark; Kolch, Walter; Waters, Katrina M.; Liu, Tao; Thrall, Brian D.; Wiley, H. S.

    2008-12-01

    Modern transcriptomics and proteomics enable us to survey the expression of RNAs and proteins at large scales. While these data are usually generated and analysed separately, there is an increasing interest in comparing and co-analysing transcriptome and proteome expression data. A major open question is whether transcriptome and proteome expression is linked and how it is coordinated. Results: Here we have developed a probabilistic clustering model that permits analysis of the links between transcriptomic and proteomic profiles in a sensible and flexible manner. Our coupled mixture model defines a prior probability distribution over the component to which a protein profile should be assigned conditioned on which component the associated mRNA profile belongs to. By providing probabilistic assignments this approach sits between the two extremes of concatenating the data on the assumption that mRNA and protein clusters would have a one-to-one relationship, and independent clustering where the mRNA profile provides no information on the protein profile and vice-versa. We apply this approach to a large dataset of quantitative transcriptomic and proteomic expression data obtained from a human breast epithelial cell line (HMEC) stimulated by epidermal growth factor (EGF) over a series of timepoints corresponding to one cell cycle. The results reveal a complex relationship between transcriptome and proteome with most mRNA clusters linked to at least two protein clusters, and vice versa. A more detailed analysis incorporating information on gene function from the gene ontology database shows that a high correlation of mRNA and protein expression is limited to the components of some molecular machines, such as the ribosome, cell adhesion complexes and the TCP-1 chaperonin involved in protein folding. Conclusions: The dynamic regulation of the transcriptome and proteome in mammalian cells in response to an acute mitogenic stimulus appears largely independent with very little

  12. ProfileDB: a resource for proteomics and cross-omics biomarker discovery.

    PubMed

    Bauer, Chris; Glintschert, Alexander; Schuchhardt, Johannes

    2014-05-01

    The increasing size and complexity of high-throughput datasets pose a growing challenge for researchers. Often very different (cross-omics) techniques with individual data analysis pipelines are employed making a unified biomarker discovery strategy and a direct comparison of different experiments difficult and time consuming. Here we present the comprehensive web-based application ProfileDB. The application is designed to integrate data from different high-throughput 'omics' data types (Transcriptomics, Proteomics, Metabolomics) with clinical parameters and prior knowledge on pathways and ontologies. Beyond data storage, ProfileDB provides a set of dedicated tools for study inspection and data visualization. The user can gain insights into a complex experiment with just a few mouse clicks. We will demonstrate the application by presenting typical use cases for the identification of proteomics biomarkers. All presented analyses can be reproduced using the public ProfileDB web server. The ProfileDB application is available by standard browser (Firefox 18+, Internet Explorer Version 9+) technology via http://profileDB.-microdiscovery.de/ (login and pass-word: profileDB). The installation contains several public datasets including different cross-'omics' experiments. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge. PMID:24270047

  13. Differential proteomic profiles from distinct Toxoplasma gondii strains revealed by 2D-difference gel electrophoresis.

    PubMed

    Zhou, Huaiyu; Zhao, Qunli; Das Singla, Lachhman; Min, Juan; He, Shenyi; Cong, Hua; Li, Ying; Su, Chunlei

    2013-04-01

    Toxoplasma gondii is an obligate intracellular protozoan that infects mammals and birds. Human infection during pregnancy may cause severe damage to the fetus. Reactivation of latent infection in immunocompromised patients can cause life-threatening encephalitis. T. gondii strains are highly diverse but only a few lineages (Type I, II and III) are widely spread. In mouse model, Type I strains are highly virulent, whereas Type II and III strains are intermediately or non virulent. It is not clear how much quantitative difference exists in proteomic profiles among these distinct T. gondii lineages. In the present study, the proteomic profiles of T. gondii tachyzoites from these lineages were investigated by two dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS) technologies. A total of 2321 protein spots were detected. Overall, the GT1 strain of Type I lineage and the strain PTG of Type II lineage have highly similar proteomic profiles and both are different from that of the CTG strain of Type III lineage. Eighty-four protein spots were differentially expressed by greater than 1.5-fold in relative abundance and 10 of them were identified to 7 T. gondii proteins in existing database. Investigation of the quantitative differences in proteomics among distinct T. gondii strains should facilitate our understanding of difference in biological processes and pathogenesis of distinct T. gondii genotypes, which will provide basic information to determine treatment regimen for different manifestation of toxoplasmosis. PMID:23340323

  14. Proteomic profiling of the infective trophozoite stage of Acanthamoeba polyphaga.

    PubMed

    Caumo, Karin Silva; Monteiro, Karina Mariante; Ott, Thiely Rodrigues; Maschio, Vinicius José; Wagner, Glauber; Ferreira, Henrique Bunselmeyer; Rott, Marilise Brittes

    2014-12-01

    Acanthamoeba polyphaga is a free-living protozoan pathogen, whose infective trophozoite form is capable of causing a blinding keratitis and fatal granulomatous encephalitis in humans. The damage caused by A. polyphaga trophozoites in human corneal or brain infections is the result of several different pathogenic mechanisms that have not yet been elucidated at the molecular level. We performed a comprehensive analysis of the proteins expressed by A. polyphaga trophozoites, based on complementary 2-DE MS/MS and gel-free LC-MS/MS approaches. Overall, 202 non-redundant proteins were identified. An A. polyphaga proteomic map in the pH range 3-10 was produced, with protein identification for 184 of 370 resolved spots, corresponding to 142 proteins. Additionally, 94 proteins were identified by gel-free LC-MS/MS. Functional classification revealed several proteins with potential importance for pathogen survival and infection of mammalian hosts, including surface proteins and proteins related to defense mechanisms. Our study provided the first comprehensive proteomic survey of the trophozoite infective stage of an Acanthamoeba species, and established foundations for prospective, comparative and functional studies of proteins involved in mechanisms of survival, development, and pathogenicity in A. polyphaga and other pathogenic amoebae. PMID:25149354

  15. Transcriptional and proteomic profiling of flatfish (Solea senegalensis) spermatogenesis.

    PubMed

    Forné, Ignasi; Castellana, Bárbara; Marín-Juez, Rubén; Cerdà, Joan; Abián, Joaquín; Planas, Josep V

    2011-06-01

    The Senegalese sole (Solea senegalensis) is a marine flatfish of high economic value and a target species for aquaculture. The efforts to reproduce this species in captivity have been hampered by the fact that farmed males (F1) often show lower sperm production and fertilization capacity than wild-type males (F0). Our knowledge on spermatogenesis is however limited to a few studies. In a previous work, we identified by 2-D DIGE several potential protein markers in testis for the poor reproductive performance of F1 males. Therefore, the objectives of the present study were, first, to investigate changes in genes and proteins expressed in the testis throughout spermatogenesis in F0 males by using a combination of transcriptomic and proteomic approaches and, second, to further compare the testis proteome between late spermatogenic stages of F0 and F1 fish to identify potential indicators of hampered reproductive performance in F1 fish. We identified approximately 400 genes and 49 proteins that are differentially expressed during the progression of spermatogenesis and that participate in processes such as transcriptional activation, the ubiquitin-proteasome system, sperm maturation and motility or cytoskeletal remodeling. Interestingly, a number of these proteins differed in abundance between F0 and F1 fish, pointing toward alterations in cytoskeleton, sperm motility, the ubiquitin-proteasome system and the redox state during spermiogenesis as possible causes for the decreased fertility of F1 fish. PMID:21538881

  16. Proteomic Profiling of Bifidobacterium bifidum S17 Cultivated Under In Vitro Conditions

    PubMed Central

    Wei, Xiao; Wang, Simiao; Zhao, Xiangna; Wang, Xuesong; Li, Huan; Lin, Weishi; Lu, Jing; Zhurina, Daria; Li, Boxing; Riedel, Christian U.; Sun, Yansong; Yuan, Jing

    2016-01-01

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

  17. Proteomic Profiling of Bifidobacterium bifidum S17 Cultivated Under In Vitro Conditions.

    PubMed

    Wei, Xiao; Wang, Simiao; Zhao, Xiangna; Wang, Xuesong; Li, Huan; Lin, Weishi; Lu, Jing; Zhurina, Daria; Li, Boxing; Riedel, Christian U; Sun, Yansong; Yuan, Jing

    2016-01-01

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

  18. Unravelling the proteomic profile of rice meiocytes during early meiosis

    PubMed Central

    Collado-Romero, Melania; Alós, Enriqueta; Prieto, Pilar

    2014-01-01

    Transfer of genetic traits from wild or related species into cultivated rice is nowadays an important aim in rice breeding. Breeders use genetic crosses to introduce desirable genes from exotic germplasms into cultivated rice varieties. However, in many hybrids there is only a low level of pairing (if existing) and recombination at early meiosis between cultivated rice and wild relative chromosomes. With the objective of getting deeper into the knowledge of the proteins involved in early meiosis, when chromosomes associate correctly in pairs and recombine, the proteome of isolated rice meiocytes has been characterized by nLC-MS/MS at every stage of early meiosis (prophase I). Up to 1316 different proteins have been identified in rice isolated meiocytes in early meiosis, being 422 exclusively identified in early prophase I (leptotene, zygotene, or pachytene). The classification of proteins in functional groups showed that 167 were related to chromatin structure and remodeling, nucleic acid binding, cell-cycle regulation, and cytoskeleton. Moreover, the putative roles of 16 proteins which have not been previously associated to meiosis or were not identified in rice before, are also discussed namely: seven proteins involved in chromosome structure and remodeling, five regulatory proteins [such as SKP1 (OSK), a putative CDK2 like effector], a protein with RNA recognition motifs, a neddylation-related protein, and two microtubule-related proteins. Revealing the proteins involved in early meiotic processes could provide a valuable tool kit to manipulate chromosome associations during meiosis in rice breeding programs. The data have been deposited to the ProteomeXchange with the PXD001058 identifier. PMID:25104955

  19. Proteome profile of salt gland-rich epidermis extracted from a salt-tolerant tree species.

    PubMed

    Tan, Wee-Kee; Ang, Yiqian; Lim, Teck-Kwang; Lim, Tit-Meng; Kumar, Prakash; Loh, Chiang-Shiong; Lin, Qingsong

    2015-10-01

    Preparation of proteins from salt-gland-rich tissues of mangrove plant is necessary for a systematic study of proteins involved in the plant's unique desalination mechanism. Extraction of high-quality proteins from the leaves of mangrove tree species, however, is difficult due to the presence of high levels of endogenous phenolic compounds. In our study, preparation of proteins from only a part of the leaf tissues (i.e. salt gland-rich epidermal layers) was required, rendering extraction even more challenging. By comparing several extraction methods, we developed a reliable procedure for obtaining proteins from salt gland-rich tissues of the mangrove species Avicennia officinalis. Protein extraction was markedly improved using a phenol-based extraction method. Greater resolution 1D protein gel profiles could be obtained. More promising proteome profiles could be obtained through 1D-LC-MS/MS. The number of proteins detected was twice as much as compared to TUTS extraction method. Focusing on proteins that were solely present in each extraction method, phenol-based extracts contained nearly ten times more proteins than those in the extracts without using phenol. The approach could thus be applied for downstream high-throughput proteomic analyses involving LC-MS/MS or equivalent. The proteomics data presented herein are available via ProteomeXchange with identifier PXD001691. PMID:26105009

  20. The distinctive gastric fluid proteome in gastric cancer reveals a multi-biomarker diagnostic profile

    PubMed Central

    Kon, Oi Lian; Yip, Tai-Tung; Ho, Meng Fatt; Chan, Weng Hoong; Wong, Wai Keong; Tan, Soo Yong; Ng, Wai Har; Kam, Siok Yuen; Eng, Alvin KH; Ho, Patrick; Viner, Rosa; Ong, Hock Soo; Kumarasinghe, M Priyanthi

    2008-01-01

    Background Overall gastric cancer survival remains poor mainly because there are no reliable methods for identifying highly curable early stage disease. Multi-protein profiling of gastric fluids, obtained from the anatomic site of pathology, could reveal diagnostic proteomic fingerprints. Methods Protein profiles were generated from gastric fluid samples of 19 gastric cancer and 36 benign gastritides patients undergoing elective, clinically-indicated gastroscopy using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry on multiple ProteinChip arrays. Proteomic features were compared by significance analysis of microarray algorithm and two-way hierarchical clustering. A second blinded sample set (24 gastric cancers and 29 clinically benign gastritides) was used for validation. Results By significance analysyis of microarray, 60 proteomic features were up-regulated and 46 were down-regulated in gastric cancer samples (p < 0.01). Multimarker clustering showed two distinctive proteomic profiles independent of age and ethnicity. Eighteen of 19 cancer samples clustered together (sensitivity 95%) while 27/36 of non-cancer samples clustered in a second group. Nine non-cancer samples that clustered with cancer samples included 5 pre-malignant lesions (1 adenomatous polyp and 4 intestinal metaplasia). Validation using a second sample set showed the sensitivity and specificity to be 88% and 93%, respectively. Positive predictive value of the combined data was 0.80. Selected peptide sequencing identified pepsinogen C and pepsin A activation peptide as significantly down-regulated and alpha-defensin as significantly up-regulated. Conclusion This simple and reproducible multimarker proteomic assay could supplement clinical gastroscopic evaluation of symptomatic patients to enhance diagnostic accuracy for gastric cancer and pre-malignant lesions. PMID:18950519

  1. Proteomic profiling of 16 cereal grains and the application of targeted proteomics to detect wheat contamination.

    PubMed

    Colgrave, Michelle L; Goswami, Hareshwar; Byrne, Keren; Blundell, Malcolm; Howitt, Crispin A; Tanner, Gregory J

    2015-06-01

    Global proteomic analysis utilizing SDS-PAGE, Western blotting and LC-MS/MS of total protein and gluten-enriched extracts derived from 16 economically important cereals was undertaken, providing a foundation for the development of MS-based quantitative methodologies that would enable the detection of wheat contamination in foods. The number of proteins identified in each grain correlated with the number of entries in publicly available databases, highlighting the importance of continued advances in genome sequencing to facilitate accurate protein identification. Subsequently, candidate wheat-specific peptide markers were evaluated by multiple-reaction monitoring MS. The selected markers were unique to wheat, yet present in a wide range of wheat varieties that represent up to 80% of the bread wheat genome. The final analytical method was rapid (15 min) and robust (CV < 10%), showed linearity (R(2) > 0.98) spanning over 3 orders of magnitude, and was highly selective and sensitive with detection down to 15 mg/kg in intentionally contaminated soy flour. Furthermore, application of this technology revealed wheat contamination in commercially sourced flours, including rye, millet, oats, sorghum, buckwheat and three varieties of soy. PMID:25873154

  2. The impact of blood on liver metabolite profiling - a combined metabolomic and proteomic approach.

    PubMed

    Ly-Verdú, Saray; Schaefer, Alexander; Kahle, Melanie; Groeger, Thomas; Neschen, Susanne; Arteaga-Salas, Jose M; Ueffing, Marius; de Angelis, Martin Hrabe; Zimmermann, Ralf

    2014-02-01

    Metabolomics has entered the well-established omic sciences as it is an indispensable information resource to achieve a global picture of biological systems. The aim of the present study was to estimate the influence of blood removal from mice liver as part of sample preparation for metabolomic and proteomic studies. For this purpose, perfused mice liver tissue (i.e. with blood removed) and unperfused mice liver tissue (i.e. containing blood) were compared by two-dimensional gas chromatography time of flight mass spectrometry (GC × GC-TOFMS) for the metabolomic part, and by liquid chromatography tandem mass spectrometry (LC-MS/MS) for the proteomic part. Our data showed significant differences between the unperfused and perfused liver tissue samples. Furthermore, we also observed an overlap of blood and tissue metabolite profiles in our data, suggesting that the perfusion of liver tissue prior to analysis is beneficial for an accurate metabolic profile of this organ. PMID:23934789

  3. Proteomic Analysis of the Protein Expression Profile in the Mature Nigella sativa (Black Seed).

    PubMed

    Alanazi, Ibrahim O; Benabdelkamel, Hicham; Alfadda, Assim A; AlYahya, Sami A; Alghamdi, Waleed M; Aljohi, Hasan A; Almalik, Abdulaziz; Masood, Afshan

    2016-08-01

    Nigella sativa (N. sativa) seed has been used as an important nutritional flavoring agent and in traditional medicine for treating many illnesses since ancient times. Understanding the proteomic component of the seed may lead to enhance the understanding of its structural and biological functional complexity. In this study, we have analyzed its proteome profile based on gel-based proteome mapping technique that includes one-dimensional gel electrophoresis followed by liquid chromatography and tandem mass spectrometry strategy. We have not come across any such studies that have been performed in N. sativa seeds up to date. A total of 277 proteins were identified, and their functional, metabolic, and location-wise annotations were carried out using the UniProt database. The majority of proteins identified in the proteome dataset based on their function were those involved in enzyme catalytic activity, nucleotide binding, and protein binding while the major cellular processes included regulation of biological process followed by regulation of secondary biological process, cell organization and biogenesis, protein metabolism, and transport. The identified proteome was localized mainly to the nucleus then to the cytoplasm, plasma membrane, mitochondria, plastid, and others. A majority of the proteins were involved in biochemical pathways involving carbohydrate metabolism, amino acid and shikimate pathway, lipid metabolism, nucleotide, cell organization and biogenesis, transport, and defense processes. The identified proteins in the dataset help to improve our understanding of the pathways involved in N. sativa seed metabolism and its biochemical features and detail out useful information that may help to utilize these proteins. This study could thus pave a way for future further high-throughput studies using a more targeted proteomic approach. PMID:27020565

  4. Trauma-associated Human Neutrophil Alterations Revealed by Comparative Proteomics Profiling

    PubMed Central

    Zhou, Jian-Ying; Krovvidi, Ravi K.; Gao, Yuqian; Gao, Hong; Petritis, Brianne O.; De, Asit; Miller-Graziano, Carol; Bankey, Paul E.; Petyuk, Vladislav A.; Nicora, Carrie D.; Clauss, Therese R; Moore, Ronald J.; Shi, Tujin; Brown, Joseph N.; Kaushal, Amit; Xiao, Wenzhong; Davis, Ronald W.; Maier, Ronald V.; Tompkins, Ronald G.; Qian, Wei-Jun; Camp, David G.; Smith, Richard D.

    2013-01-01

    PURPOSE Polymorphonuclear neutrophils (PMNs) play an important role in mediating the innate immune response after severe traumatic injury; however, the cellular proteome response to traumatic condition is still largely unknown. EXPERIMENTAL DESIGN We applied 2D-LC-MS/MS based shotgun proteomics to perform comparative proteome profiling of human PMNs from severe trauma patients and healthy controls. RESULTS A total of 197 out of ~2500 proteins (being identified with at least two peptides) were observed with significant abundance changes following the injury. The proteomics data were further compared with transcriptomics data for the same genes obtained from an independent patient cohort. The comparison showed that the protein abundance changes for the majority of proteins were consistent with the mRNA abundance changes in terms of directions of changes. Moreover, increased protein secretion was suggested as one of the mechanisms contributing to the observed discrepancy between protein and mRNA abundance changes. Functional analyses of the altered proteins showed that many of these proteins were involved in immune response, protein biosynthesis, protein transport, NRF2-mediated oxidative stress response, the ubiquitin-proteasome system, and apoptosis pathways. CONCLUSIONS AND CLINICAL RELEVANCE Our data suggest increased neutrophil activation and inhibited neutrophil apoptosis in response to trauma. The study not only reveals an overall picture of functional neutrophil response to trauma at the proteome level, but also provides a rich proteomics data resource of trauma-associated changes in the neutrophil that will be valuable for further studies of the functions of individual proteins in PMNs. PMID:23589343

  5. Cigarette smoke alters the proteomic profile of lung fibroblasts.

    PubMed

    D'Anna, Claudia; Cigna, Diego; Costanzo, Giorgia; Bruno, Andreina; Ferraro, Maria; Di Vincenzo, Serena; Bianchi, Laura; Bini, Luca; Gjomarkaj, Mark; Pace, Elisabetta

    2015-06-01

    Smoking is strongly associated with diseases such as lung cancer and chronic obstructive pulmonary disease (COPD). Lung fibroblasts are crucial for the integrity of alveolar structure by producing extracellular matrix proteins which are required for attachment, structure, and function of alveolar epithelial cells. Despite the well-known association between cigarette smoke exposure and pulmonary and cardiovascular diseases, many questions remain regarding the mechanisms by which smoking induces diseases. The aim of this study is to detect differentially expressed proteins in human foetal lung cells (HFL-1) after 5 and 10% doses of cigarette smoke extract (CSE) exposure, combining two-dimensional electrophoresis (2DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In order to evaluate cellular ability to recover as well as lasting damage, we analysed the proteomic pattern 24 hours after the CSE removal (release). Eleven proteins had significant changes at various experimental points. Among these, 7 were up-regulated after CSE-treatments and 4 were down-regulated. Some spots seemed to be modified permanently or in a transient manner, in fact they returned to baseline levels after CSE-removal (normalisation after CSE release) and others were modified by selective CSE concentrations or only after release. MS identified, differentially expressed proteins are involved in stress response, mitochondrial activity, and aging. These findings may improve our understanding about molecular mechanisms underlying CSE caused damage and they may also integrate the comprehension of cigarette smoke effects on human health. PMID:25900673

  6. Proteomic Profiling of Mouse Liver following Acute Toxoplasma gondii Infection.

    PubMed

    He, Jun-Jun; Ma, Jun; Elsheikha, Hany M; Song, Hui-Qun; Zhou, Dong-Hui; Zhu, Xing-Quan

    2016-01-01

    Toxoplasma gondii remains a global public health problem. However, its pathophysiology is still not-completely understood particularly the impact of infection on host liver metabolism. We performed iTRAQ-based proteomic analysis to evaluate early liver protein responses in BALB/c mice following infection with T. gondii PYS strain (genotype ToxoDB#9) infection. Our data revealed modification of protein expression in key metabolic pathways, as indicated by the upregulation of immune response and downregulation of mitochondrial respiratory chain, and the metabolism of fatty acids, lipids and xenobiotics. T. gondii seems to hijack host PPAR signaling pathway to downregulate the metabolism of fatty acids, lipids and energy in the liver. The metabolism of over 400 substances was affected by the downregulation of genes involved in xenobiotic metabolism. The top 10 transcription factors used by upregulated genes were Stat2, Stat1, Irf2, Irf1, Sp2, Egr1, Stat3, Klf4, Elf1 and Gabpa, while the top 10 transcription factors of downregulated genes were Hnf4A, Ewsr1, Fli1, Hnf4g, Nr2f1, Pparg, Rxra, Hnf1A, Foxa1 and Foxo1. These findings indicate global reprogramming of the metabolism of the mouse liver after acute T. gondii infection. Functional characterization of the altered proteins may enhance understanding of the host responses to T. gondii infection and lead to the identification of new therapeutic targets. PMID:27003162

  7. Proteomic Profiling of Mouse Liver following Acute Toxoplasma gondii Infection

    PubMed Central

    He, Jun-Jun; Ma, Jun; Elsheikha, Hany M.; Song, Hui-Qun; Zhou, Dong-Hui; Zhu, Xing-Quan

    2016-01-01

    Toxoplasma gondii remains a global public health problem. However, its pathophysiology is still not-completely understood particularly the impact of infection on host liver metabolism. We performed iTRAQ-based proteomic analysis to evaluate early liver protein responses in BALB/c mice following infection with T. gondii PYS strain (genotype ToxoDB#9) infection. Our data revealed modification of protein expression in key metabolic pathways, as indicated by the upregulation of immune response and downregulation of mitochondrial respiratory chain, and the metabolism of fatty acids, lipids and xenobiotics. T. gondii seems to hijack host PPAR signaling pathway to downregulate the metabolism of fatty acids, lipids and energy in the liver. The metabolism of over 400 substances was affected by the downregulation of genes involved in xenobiotic metabolism. The top 10 transcription factors used by upregulated genes were Stat2, Stat1, Irf2, Irf1, Sp2, Egr1, Stat3, Klf4, Elf1 and Gabpa, while the top 10 transcription factors of downregulated genes were Hnf4A, Ewsr1, Fli1, Hnf4g, Nr2f1, Pparg, Rxra, Hnf1A, Foxa1 and Foxo1. These findings indicate global reprogramming of the metabolism of the mouse liver after acute T. gondii infection. Functional characterization of the altered proteins may enhance understanding of the host responses to T. gondii infection and lead to the identification of new therapeutic targets. PMID:27003162

  8. Proteomic profiling of lymphedema development in mouse model.

    PubMed

    Lee, Joomin; Song, Haeun; Roh, Kangsan; Cho, Sungrae; Lee, Sukchan; Yeom, Chang-Hwan; Park, Seyeon

    2016-07-01

    The lymphatic vascular system plays an important role in tissue fluid homeostasis. Lymphedema is a chronic, progressive, and incurable condition that leads to lymphatic fluid retention; it may be primary (heritable) or secondary (acquired) in nature. Although there is a growing understanding of lymphedema, methods for the prevention and treatment of lymphedema are still limited. In this study, we investigated differential protein expressions in sham-operated and lymphedema-operated mice for 3 days, using two-dimensional gel electrophoresis (2-DE) and mass spectrometry analysis. Male improved methodology for culturing noninbred (ICR) mice developed lymphedema in the right hindlimb. Twenty functional proteins were found to be differentially expressed between lymphedema induced-right leg tissue and normal left leg tissue. Out of these proteins, the protein levels of apolipoprotein A-1 preprotein, alpha-actinin-3, mCG21744, parkinson disease, serum amyloid P-component precursor, annexin A8, mKIAA0098 protein, and fibrinogen beta chain precursor were differentially upregulated in the lymphedema mice compared with the sham-operated group. Western blotting analysis was used to validate the proteomics results. Our results showing differential up-regulation of serum amyloid P-component precursor, parkinson disease, and apolipoprotein A-1 preprotein in lymphedema model over sham-operated model suggest important insights into pathophysiological target for lymphedema. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27151289

  9. Multilineage potential and proteomic profiling of human dental stem cells derived from a single donor

    SciTech Connect

    Patil, Rajreddy; Kumar, B. Mohana; Lee, Won-Jae; Jeon, Ryoung-Hoon; Jang, Si-Jung; Lee, Yeon-Mi; Park, Bong-Wook; Byun, June-Ho; Ahn, Chun-Seob; Kim, Jae-Won; Rho, Gyu-Jin

    2014-01-01

    Dental tissues provide an alternative autologous source of mesenchymal stem cells (MSCs) for regenerative medicine. In this study, we isolated human dental MSCs of follicle, pulp and papilla tissue from a single donor tooth after impacted third molar extraction by excluding the individual differences. We then compared the morphology, proliferation rate, expression of MSC-specific and pluripotency markers, and in vitro differentiation ability into osteoblasts, adipocytes, chondrocytes and functional hepatocyte-like cells (HLCs). Finally, we analyzed the protein expression profiles of undifferentiated dental MSCs using 2DE coupled with MALDI-TOF-MS. Three types of dental MSCs largely shared similar morphology, proliferation potential, expression of surface markers and pluripotent transcription factors, and differentiation ability into osteoblasts, adipocytes, and chondrocytes. Upon hepatogenic induction, all MSCs were transdifferentiated into functional HLCs, and acquired hepatocyte functions by showing their ability for glycogen storage and urea production. Based on the proteome profiling results, we identified nineteen proteins either found commonly or differentially expressed among the three types of dental MSCs. In conclusion, three kinds of dental MSCs from a single donor tooth possessed largely similar cellular properties and multilineage potential. Further, these dental MSCs had similar proteomic profiles, suggesting their interchangeable applications for basic research and call therapy. - Highlights: • Isolated and characterized three types of human dental MSCs from a single donor. • MSCs of dental follicle, pulp and papilla had largely similar biological properties. • All MSCs were capable of transdifferentiating into functional hepatocyte-like cells. • 2DE proteomics with MALDI-TOF/MS identified 19 proteins in three types of MSCs. • Similar proteomic profiles suggest interchangeable applications of dental MSCs.

  10. Proteomic profiling of serum samples from chikungunya-infected patients provides insights into host response

    PubMed Central

    2013-01-01

    Background Chikungunya is a highly debilitating febrile illness caused by Chikungunya virus, a single-stranded RNA virus, which is transmitted by Aedes aegypti or Aedes albopictus mosquito species. The pathogenesis and host responses in individuals infected with the chikungunya virus are not well understood at the molecular level. We carried out proteomic profiling of serum samples from chikungunya patients in order to identify molecules associated with the host response to infection by this virus. Results Proteomic profiling of serum obtained from the infected individuals resulted in identification of 569 proteins. Of these, 63 proteins were found to be differentially expressed (≥ 2-fold) in patient as compared to control sera. These differentially expressed proteins were involved in various processes such as lipid metabolism, immune response, transport, signal transduction and apoptosis. Conclusions This is the first report providing a global proteomic profile of serum samples from individuals infected with the chikungunya virus. Our data provide an insight into the proteins that are involved as host response factors during an infection. These proteins include clusterin, apolipoproteins and S100A family of proteins. PMID:24124767

  11. Proteomic Analysis of Propiconazole Responses in Mouse Liver-Comparison of Genomic and Proteomic Profiles

    EPA Science Inventory

    We have performed for the first time a comprehensive profiling of changes in protein expression of soluble proteins in livers from mice treated with the mouse liver tumorigen, propiconazole, to uncover the pathways and networks altered by this commonly used fungicide. Utilizing t...

  12. Proteomic analysis of propiconazole responses in mouse liver: comparison of genomic and proteomic profiles

    EPA Science Inventory

    We have performed for the first time a comprehensive profiling of changes in protein expression of soluble proteins in livers from mice treated with the mouse liver tumorigen, propiconazole, to uncover the pathways and networks altered by this fungicide. Utilizing twodimensional...

  13. Proteomic profiling of salivary gland after nonviral gene transfer mediated by conventional plasmids and minicircles

    PubMed Central

    Geguchadze, Ramaz; Wang, Zhimin; Zourelias, Lee; Perez-Riveros, Paola; Edwards, Paul C; Machen, Laurie; Passineau, Michael J

    2014-01-01

    In this study, we compared gene transfer efficiency and host response to ultrasound-assisted, nonviral gene transfer with a conventional plasmid and a minicircle vector in the submandibular salivary glands of mice. Initially, we looked at gene transfer efficiency with equimolar amounts of the plasmid and minicircle vectors, corroborating an earlier report showing that minicircle is more efficient in the context of a physical method of gene transfer. We then sought to characterize the physiological response of the salivary gland to exogenous gene transfer using global proteomic profiling. Somewhat surprisingly, we found that sonoporation alone, without a gene transfer vector present, had virtually no effect on the salivary gland proteome. However, when a plasmid vector was used, we observed profound perturbations of the salivary gland proteome that compared in magnitude to that seen in a previous report after high doses of adeno-associated virus. Finally, we found that gene transfer with a minicircle induces only minor proteomic alterations that were similar to sonoporation alone. Using mass spectrometry, we assigned protein IDs to 218 gel spots that differed between plasmid and minicircle. Bioinformatic analysis of these proteins demonstrated convergence on 68 known protein interaction pathways, most notably those associated with innate immunity, cellular stress, and morphogenesis. PMID:25414909

  14. Novel possibilities in the study of the salivary proteomic profile using SELDI-TOF/MS technology

    PubMed Central

    ARDITO, FATIMA; PERRONE, DONATELLA; COCCHI, ROBERTO; LO RUSSO, LUCIO; DE LILLO, ALFREDO; GIANNATEMPO, GIOVANNI; LO MUZIO, LORENZO

    2016-01-01

    There is currently an increasing interest in exploring human saliva to identify salivary diagnostic and prognostic biomarkers, since the collection of saliva is rapid, non-invasive and stress-free. Diagnostic tests on saliva are common and cost-effective, particularly for patients who need to monitor their hormone levels or the effectiveness of undergoing therapies. Furthermore, salivary diagnostics is ideal for surveillance studies and in situations where fast results and inexpensive technologies are required. The most important constituents of saliva are proteins, the expression levels of which may be modified due to variations of the cellular conditions. Therefore, the different profile of proteins detected in saliva, including their absence, presence or altered levels, is a potential biomarker of certain physiological and/or pathological conditions. A promising novel approach to study saliva is the global analysis of salivary proteins using proteomic techniques. In the present study, surface-enhanced laser desorption/ionization-time-of-flight/mass spectrometry (SELDI-TOF/MS), one of the most recent proteomic tools for the identification of novel biomarkers, is reviewed. In addition, the possible use of this technique in salivary proteomic studies is discussed, since SELDI technology combines the precision of matrix-assisted laser desorption/ionization-TOF/MS proteomic analysis and the high-throughput nature of protein array analysis. PMID:26998108

  15. Proteomic Profiles of Mesenchymal Stem Cells Induced by a Liver Differentiation Protocol

    PubMed Central

    Leelawat, Kawin; Narong, Siriluck; Chaijan, Suthidarak; Sa-ngiamsuntorn, Khanit; Disthabanchong, Sinee; Wongkajornsilp, Adisak; Hongeng, Suradej

    2010-01-01

    The replacement of disease hepatocytes and the stimulation of endogenous or exogenous regeneration by human mesenchymal stem cells (MSCs) are promising candidates for liver-directed cell therapy. In this study, we isolated MSCs from adult bone marrow by plastic adhesion and induced differentiation with a liver differentiation protocol. Western blot analyses were used to assess the expression of liver-specific markers. Next, MSC-specific proteins were analyzed with two-dimensional (2D) gel electrophoresis and peptide mass fingerprinting matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS). To confirm the results from the proteomic study, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed. We demonstrated that MSCs treated with the liver differentiation protocol expressed significantly more albumin, CK19 and CK20, than did undifferentiated cells. In addition the results of proteomic study demonstrated increases expression of FEM1B, PSMC2 and disulfide-isomerase A3 in MSCs treated with the liver differentiation protocol. These results from proteomic profiling will not only provide insight into the global responses of MSCs to hepatocyte differentiation, but will also lead to in-depth studies on the mechanisms of proteomic changes in MSCs. PMID:21614181

  16. Gender Specific Differences in the Perceived Antecedents of Academic Stress.

    ERIC Educational Resources Information Center

    Jones, Russell W.

    This document consists of the report of a study undertaken to establish the existence of any gender specific differences in the perceived antecedents of academic stress. The definition of stress as a negative emotion strongly associated with doubt about coping is suggested to be particularly relevant to the academic arena where students…

  17. Smoking among Dutch Elementary Schoolchildren: Gender-Specific Predictors

    ERIC Educational Resources Information Center

    Ausems, M.; Mesters, I.; van Breukelen, G.; De Vries, H.

    2009-01-01

    Higher rates of smoking initiation and continuation by female compared with male adolescents, as found in many developed countries, may call for gender-specific prevention programs. Risk factors of smoking initiation and continuation were examined prospectively (1997-2002) among 3205 Dutch elementary schoolchildren (mean age 11.64) in an…

  18. Profiling thiol redox proteome using isotope tagging mass spectrometry.

    PubMed

    Parker, Jennifer; Zhu, Ning; Zhu, Mengmeng; Chen, Sixue

    2012-01-01

    Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant(1,2). The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato(3). Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas(3). Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)(4). DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue(4). ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins(5). Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes(6,7). Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry(8,9). The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter(10). After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic

  19. Elucidation of Xenobiotic Metabolism Pathways in Human Skin and Human Skin Models by Proteomic Profiling

    PubMed Central

    van Eijl, Sven; Zhu, Zheying; Cupitt, John; Gierula, Magdalena; Götz, Christine; Fritsche, Ellen; Edwards, Robert J.

    2012-01-01

    Background Human skin has the capacity to metabolise foreign chemicals (xenobiotics), but knowledge of the various enzymes involved is incomplete. A broad-based unbiased proteomics approach was used to describe the profile of xenobiotic metabolising enzymes present in human skin and hence indicate principal routes of metabolism of xenobiotic compounds. Several in vitro models of human skin have been developed for the purpose of safety assessment of chemicals. The suitability of these epidermal models for studies involving biotransformation was assessed by comparing their profiles of xenobiotic metabolising enzymes with those of human skin. Methodology/Principal Findings Label-free proteomic analysis of whole human skin (10 donors) was applied and analysed using custom-built PROTSIFT software. The results showed the presence of enzymes with a capacity for the metabolism of alcohols through dehydrogenation, aldehydes through dehydrogenation and oxidation, amines through oxidation, carbonyls through reduction, epoxides and carboxylesters through hydrolysis and, of many compounds, by conjugation to glutathione. Whereas protein levels of these enzymes in skin were mostly just 4–10 fold lower than those in liver and sufficient to support metabolism, the levels of cytochrome P450 enzymes were at least 300-fold lower indicating they play no significant role. Four epidermal models of human skin had profiles very similar to one another and these overlapped substantially with that of whole skin. Conclusions/Significance The proteomics profiling approach was successful in producing a comprehensive analysis of the biotransformation characteristics of whole human skin and various in vitro skin models. The results show that skin contains a range of defined enzymes capable of metabolising different classes of chemicals. The degree of similarity of the profiles of the in vitro models indicates their suitability for epidermal toxicity testing. Overall, these results provide a

  20. Statistical and computational methods for comparative proteomic profiling using liquid chromatography-tandem mass spectrometry.

    PubMed

    Listgarten, Jennifer; Emili, Andrew

    2005-04-01

    The combined method of LC-MS/MS is increasingly being used to explore differences in the proteomic composition of complex biological systems. The reliability and utility of such comparative protein expression profiling studies is critically dependent on an accurate and rigorous assessment of quantitative changes in the relative abundance of the myriad of proteins typically present in a biological sample such as blood or tissue. In this review, we provide an overview of key statistical and computational issues relevant to bottom-up shotgun global proteomic analysis, with an emphasis on methods that can be applied to improve the dependability of biological inferences drawn from large proteomic datasets. Focusing on a start-to-finish approach, we address the following topics: 1) low-level data processing steps, such as formation of a data matrix, filtering, and baseline subtraction to minimize noise, 2) mid-level processing steps, such as data normalization, alignment in time, peak detection, peak quantification, peak matching, and error models, to facilitate profile comparisons; and, 3) high-level processing steps such as sample classification and biomarker discovery, and related topics such as significance testing, multiple testing, and choice of feature space. We report on approaches that have recently been developed for these steps, discussing their merits and limitations, and propose areas deserving of further research. PMID:15741312

  1. Temporal proteomic profiling of Chlamydia trachomatis-infected HeLa-229 human cervical epithelial cells.

    PubMed

    Tan, Grace Min Yi; Lim, Hui Jing; Yeow, Tee Cian; Movahed, Elaheh; Looi, Chung Yeng; Gupta, Rishein; Arulanandam, Bernard P; Abu Bakar, Sazaly; Sabet, Negar Shafiei; Chang, Li-Yen; Wong, Won Fen

    2016-05-01

    Chlamydia trachomatis is the leading causative agent of bacterial sexually transmitted infections worldwide which can lead to female pelvic inflammatory disease and infertility. A greater understanding of host response during chlamydial infection is essential to design intervention technique to reduce the increasing incidence rate of genital chlamydial infection. In this study, we investigated proteome changes in epithelial cells during C. trachomatis infection by using an isobaric tags for relative and absolute quantitation (iTRAQ) labeling technique coupled with a liquid chromatography-tandem mass spectrometry (LC-MS(3) ) analysis. C. trachomatis (serovar D, MOI 1)-infected HeLa-229 human cervical carcinoma epithelial cells (at 2, 4 and 8 h) showed profound modifications of proteome profile which involved 606 host proteins. MGST1, SUGP2 and ATXN10 were among the top in the list of the differentially upregulated protein. Through pathway analysis, we suggested the involvement of eukaryotic initiation factor 2 (eIF2) and mammalian target of rapamycin (mTOR) in host cells upon C. trachomatis infection. Network analysis underscored the participation of DNA repair mechanism during C. trachomatis infection. In summary, intense modifications of proteome profile in C. trachomatis-infected HeLa-229 cells indicate complex host-pathogen interactions at early phase of chlamydial infection. PMID:27134121

  2. Proteomic profile of saliva and plasma from women with impalpable breast lesions

    PubMed Central

    Delmonico, Lucas; Bravo, Maryah; Silvestre, Rafaele Tavares; Ornellas, Maria Helena Faria; De Azevedo, Carolina Maria; Alves, Gilda

    2016-01-01

    The present study evaluated the proteomic profile of saliva and plasma from women with impalpable breast lesions using nano-liquid chromatography-quadrupole-time-of-flight (nLC-Q-TOF) technology. Plasma and saliva from patients with fibroadenoma (n=10), infiltrating ductal carcinoma (n=10) and healthy control groups (n=8) were assessed by combinations of inter/intra-group analyses, revealing significant quantitative and qualitative differences. The major differentially-expressed proteins in the saliva of patients compared with the controls were α2-macroglobulin and ceruloplasmin, but the proteins that met the minimum fold-change and P-value cut-offs were leukocyte elastase inhibitor and α-enolase, and deleted in malignant brain tumors 1. Concerning plasma, α-2-macroglobulin and ceruplasmin were upregulated, while other proteins such as haptoglobin, hemopexin and vitamin D-binding protein were downregulated compared with the control. The changes in immune, molecular transport and signaling pathways were the most representative in the proteomic profile of the saliva and plasma. This is the first study to describe the proteome of saliva and plasma from the same women with impalpable breast lesions. PMID:27602154

  3. Dynamic proteomic profiling of a unicellular cyanobacterium Cyanothece ATCC51142 across light-dark diurnal cycles

    SciTech Connect

    Aryal, Uma K.; Stockel, Jana; Krovvidi, Ravi K.; Gritsenko, Marina A.; Monroe, Matthew E.; Moore, Ronald J.; Koppenaal, David W.; Smith, Richard D.; Pakrasi, Himadri B.; Jacobs, Jon M.

    2011-12-01

    Unicellular cyanobacteria of the genus Cyanothece are recognized for their ability to execute nitrogen (N2)-fixation in the dark and photosynthesis in the light. Systems-wide dynamic proteomic profiling with mass spectrometry (MS) analysis reveals fundamental insights into the control and regulation of these functions. To expand upon the current knowledge of protein expression patterns in Cyanothece ATCC51142, we performed quantitative proteomic analysis using partial ("unsaturated") metabolic labeling and high mass accuracy LC-MS analysis. This dynamic proteomic profiling identified 721 actively synthesized proteins with significant temporal changes in expression throughout the light-dark cycles, of which 425 proteins matched with previously characterized cycling transcripts. The remaining 296 proteins contained a cluster of proteins uniquely involved in DNA replication and repair, protein degradation, tRNA synthesis and modification, transport and binding, and regulatory functions. Analysis of protein functions revealed that the expression of nitrogenase in the dark is mediated by higher respiration and glycogen metabolism. We have also shown that Cyanothece ATCC51142 utilizes alternative pathways for carbon (C) and nitrogen (N) acquisition, particularly, aspartic acid and glutamate as substrates of C and N, respectively. Utilization of phosphoketolase (PHK) pathway for the conversion of xylulose-5P to pyruvate and acetyl-P likely constitutes an alternative strategy to compensate higher ATP and NADPH demand. In conclusion, this study provides a deeper insight into how Cyanothece ATCC51142 modulates cellular functions to accommodate photosynthesis and N2-fixation within the single cell.

  4. Urinary proteomic profiling in severe obesity and obstructive sleep apnoea with CPAP treatment

    PubMed Central

    Seetho, Ian W; Ramírez-Torres, Adela; Albalat, Amaya; Mullen, William; Mischak, Harald; Parker, Robert J; Craig, Sonya; Duffy, Nick; Hardy, Kevin J; Burniston, Jatin G; Wilding, John PH

    2015-01-01

    Introduction Obstructive sleep apnoea (OSA) is common in obesity and is associated with cardiovascular and metabolic complications. Continuous positive airway pressure (CPAP) in OSA may lead to physiological changes reflected in the urinary proteome. The aim of this study was to characterise the urinary proteome in severely obese adult subjects with OSA who were receiving CPAP compared with severely obese subjects without OSA. Methods Severely obese subjects with and without OSA were recruited. Subjects with OSA were receiving CPAP. Body composition and blood pressure measurements were recorded. Urinary samples were analysed by Capillary Electrophoresis–Mass Spectrometry (CE–MS). Results Twenty-seven subjects with OSA-on-CPAP (age 49±7years, BMI 43±7 kg/m2) and 25 controls without OSA (age 52±9years, BMI 39±4 kg/m2) were studied. Age and BMI were not significantly different between groups. Mean CPAP use for OSA patients was 14.5±1.0 months. Metabolic syndrome was present in 14(52%) of those with OSA compared with 6(24%) of controls (p=0.039). A urinary proteome comprising 15 peptides was identified showing differential expression between the groups (p<0.01). Although correction for multiple testing did not reach significance, sequences were determined for 8 peptides demonstrating origins from collagens, fibrinogen beta chain and T-cadherin that may be associated with underlying cardiovascular disease mechanisms in OSA. Conclusions The urinary proteome is compared in OSA with CPAP and without OSA in severe obesity. The effects of CPAP on OSA may lead to changes in the urinary peptides but further research work is needed to investigate the potential role for urinary proteomics in characterising urinary peptide profiles in OSA. PMID:26483946

  5. Proteomic Profiling of Human Liver Biopsies: Hepatitis C Virus-Induced Fibrosis and Mitochondrial Dysfunction

    SciTech Connect

    Diamond, Deborah L.; Jacobs, Jon M.; Paeper, Bryan; Proll, Sean; Gritsenko, Marina A.; Carithers, Jr., Robert L.; Larson , Anne M.; Yeh, Matthew M.; Camp, David G.; Smith, Richard D.; Katze, Michael G.

    2007-09-01

    Liver biopsies from HCV-infected patients offer the unique opportunity to study human liver biology and disease in vivo. However, the low protein yields associated with these small samples present a significant challenge for proteomic analysis. In this study we describe the application of an ultra-sensitive proteomics platform for performing robust quantitative proteomic studies on microgram amounts of HCV-infected human liver tissue from 15 patients at different stages of fibrosis. A high quality liver protein data base containing 5,920 unique protein identifications supported high throughput quantitative studies using 16O:18O stable isotope labeling in combination with the accurate mass and time (AMT) tag approach. A total of 1,641 liver biopsy proteins were quantified and ANOVA identified 210 proteins exhibiting statistically significant differences associated with fibrosis stage. Hierarchical clustering revealed that biopsies representative of later fibrosis stages (e.g. Batts-Ludwig stages 3-4) exhibited a distinct protein expression profile indicating an apparent down-regulation of many proteins when compared to samples from earlier fibrosis stages (e.g. Batts-Ludwig stages 0-2). Functional analysis of these signature proteins suggests that impairment of key mitochondrial processes including fatty acid oxidation and oxidative phosphorylation, and response to oxidative stress and reactive oxygen species occurs during advanced stage 3-4 fibrosis. In conclusion, the results reported here represent a significant advancement in clinical proteomics providing to our knowledge, the first demonstration of global proteomic alterations accompanying liver disease progression in patients chronically infected with HCV. Our findings contribute to a generally emerging theme associating oxidative stress and hepatic mitochondrial dysfunction with HCV pathogenesis.

  6. O-GlcNAc profiling: from proteins to proteomes

    PubMed Central

    2014-01-01

    O-linked β-D-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) onto serine and threonine residues of proteins is an important post-translational modification (PTM), which is involved in many crucial biological processes including transcription, translation, proteasomal degradation, and signal transduction. Aberrant protein O-GlcNAcylation is directly linked to the pathological progression of chronic diseases including diabetes, cancer, and neurodegenerative disorders. Identification, site mapping, and quantification of O-GlcNAc proteins are a prerequisite to decipher their functions. In this review, we mainly focus on technological developments regarding O-GlcNAc protein profiling. Specifically, on one hand, we show how these techniques are being used for the comprehensive characterization of certain targeted proteins in which biologists are most interested. On the other hand, we present several newly developed approaches for O-GlcNAcomic profiling as well as how they provide us with a systems perspective to crosstalk amongst different PTMs and complicated biological events. Promising technical trends are also highlighted to evoke more efforts by diverse laboratories, which would further expand our understanding of the physiological and pathological roles of protein O-GlcNAcylation in chronic diseases. PMID:24593906

  7. Proteomic profile of Ortleppascaris sp.: A helminth parasite of Rhinella marina in the Amazonian region

    PubMed Central

    e Silva, Jefferson Pereira; Furtado, Adriano Penha; dos Santos, Jeannie Nascimento

    2014-01-01

    Ortleppascaris sp. is a helminth that, in its larval stage, infects the liver parenchyma of the amphibian Rhinella marina, resulting in severe physiological and pathological changes. This study used a proteomic approach to determine the overall profile of proteins expressed in a somatic extract from the nematodes to investigate the relationship between the parasite and its host. A total of 60 abundant proteins were selected from the two-dimensional electrophoresis, identified by peptide mass fingerprinting, and grouped based on their Gene Ontology by the biological processes in which they are potentially involved. Important helminthic derivatives, such as the immunoreactive As37 antigen, guanylyl cyclases, proteolytic enzymes, and other proteins conserved among different parasites, were identified through homology. This study represents a new approach to helminth-related proteomic studies using an amphibian animal model. Furthermore, this study identified protein markers that are important to the host–parasite relationship and the viability, development, infectivity, and virulence of helminths. PMID:25161903

  8. Proteomic Profiling Of Two-Dimensional Gel Electrophoresis Protein Expression Data

    NASA Astrophysics Data System (ADS)

    Ahmad, Norhaiza; Zhang, J.; Brown, P. J.; James, D. C.; Birch, J. R.; Racher, A. J.; Smales, C. M.

    2008-01-01

    We have undertaken two-dimensional gel electrophoresis (2-DE) proteomic profiling on a series of cell lines with different recombinant antibody production rates. Due to the nature of 2-DE proteomic investigations there will always be `process variability' factors in any data set collected in this way. Some of this variation will arise during sample preparation, gel running and staining, while further variation will arise from the gel analysis procedure. Therefore, in order to identify all significant changes in protein expression between biological samples when analysed by 2-DE, the system precision or `error', and how this correlates to protein abundance, must be known. Only then can the system be considered robust and investigators accurately and confidently report all observable statistically significant changes in protein expression. We introduce an expression variability test to identify protein spots whose expression correlates with increased antibody production. The results have highlighted a small number of candidate proteins for further investigation.

  9. Proteomic profiling of the influence of iron availability on Cryptococcus gattii

    PubMed Central

    Crestani, Juliana; Carvalho, Paulo Costa; Han, Xuemei; Seixas, Adriana; Broetto, Leonardo; de Saldanha da Gama Fischer, Juliana; Staats, Charley Christian; Schrank, Augusto; Yates, John R; Vainstein, Marilene Henning

    2011-01-01

    Iron is essential and ubiquitous in living organisms. The competition for this micronutrient between the host and its pathogens has been related to disease establishment. Cryptococcus gattii is an encapsulated yeast that causes cryptococcosis mainly in immunocompetent individuals. In this study, we analyzed the proteomic profile of the C. gattii R265 Vancouver Island isolate under iron-depleted and –replete conditions by Multidimensional Protein Identification Technology (MudPIT) and by 2D-GE. Proteins and key mechanisms affected by alteration of iron levels such as capsule production, cAMP-signaling pathway, response to stress, and metabolic pathways related to mitochondrial function were identified. Our results also show both proteomic methodologies employed to be complementary. PMID:21970549

  10. Profiling of Multiple Targets of Artemisinin Activated by Hemin in Cancer Cell Proteome.

    PubMed

    Zhou, Yiqing; Li, Weichao; Xiao, Youli

    2016-04-15

    The antimalarial drug artemisinin is found to have diverse biological activities ranging from anti-inflammatory to anticancer properties; however, as of today, the cellular targets and mechanism of action of this important compound have remained elusive. Here, we report the global protein target profiling of artemisinin in the HeLa cancer cell proteome using a chemical proteomics approach. In the presence of hemin, multiple proteins were targeted by artemisinin probe through covalent modification. Further studies revealed that reducing of hemin to heme by protein thiols was essential for endoperoxide activation and subsequent protein alkylation. Artemisinin may exert its synergistic therapeutic anticancer effects via modulation of a variety of cellular pathways through acting on multiple targets. PMID:26854499

  11. Proteomic Mucin Profiling for the Identification of Cystic Precursors of Pancreatic Cancer

    PubMed Central

    2014-01-01

    Background Pancreatic cystic lesions (PCLs) are increasingly frequent radiological incidentalomas, with a considerable proportion representing precursors of pancreatic cancer. Better diagnostic tools are required for patients to benefit from this development. Methods To evaluate whether cyst fluid mucin expression could predict malignant potential and/or transformation in PCLs, a proteomic method was devised and prospectively evaluated in consecutive patients referred to our tertiary center for endoscopic ultrasound-guided aspiration of cystic lesions from May 2007 through November 2008 (discovery cohort) and from December 2008 through October 2012 (validation cohort). Cytology and cyst fluid carcinoembryonic antigen (CEA; premalignancy > 192ng/mL, malignancy > 1000ng/mL) were routinely analyzed, and samples were further processed as follows: one-dimensional gel electrophoresis, excision of high-mass areas, tryptic digestion and nano-liquid chromatography–tandem mass spectrometry, with peptide identification by Mascot software and an in-house mucin database. All diagnostic evaluations were blinded to proteomics results. Histology was required to confirm the presence/absence of malignant transformation. All statistical tests were two-sided. Results Proteomic mucin profiling proved statistically significantly more accurate (97.5%; 95% confidence interval [CI] = 90.3% to 99.6%) than cytology (71.4%; 95% CI = 59.8% to 80.9%; P < .001) and cyst fluid CEA (78.0%; 95% CI = 65.0% to 87.3%; P < .001) in identifying the 37 (out of 79; 46.8%) lesions with malignant potential (ie, premalignant or malignant tumors). The accuracy of proteomics was nearly identical (96.6% vs 98.0%) between the discovery (n = 29) and validation (n = 50) cohorts. Furthermore, mucin profiling predicted malignant transformation, present in 16 out of 29 (discovery cohort: 9, validation cohort: 20) lesions with available histology, with 89.7% accuracy (95% CI = 71.5% to 97.3%) (for the validation

  12. Biomarker discovery for inflammatory bowel disease, using proteomic serum profiling.

    PubMed

    Meuwis, Marie-Alice; Fillet, Marianne; Geurts, Pierre; de Seny, Dominique; Lutteri, Laurence; Chapelle, Jean-Paul; Bours, Vincent; Wehenkel, Louis; Belaiche, Jacques; Malaise, Michel; Louis, Edouard; Merville, Marie-Paule

    2007-05-01

    Crohn's disease and ulcerative colitis known as inflammatory bowel diseases (IBD) are chronic immuno-inflammatory pathologies of the gastrointestinal tract. These diseases are multifactorial, polygenic and of unknown etiology. Clinical presentation is non-specific and diagnosis is based on clinical, endoscopic, radiological and histological criteria. Novel markers are needed to improve early diagnosis and classification of these pathologies. We performed a study with 120 serum samples collected from patients classified in 4 groups (30 Crohn, 30 ulcerative colitis, 30 inflammatory controls and 30 healthy controls) according to accredited criteria. We compared protein sera profiles obtained with a Surface Enhanced Laser Desorption Ionization-Time of Flight-Mass Spectrometer (SELDI-TOF-MS). Data analysis with univariate process and a multivariate statistical method based on multiple decision trees algorithms allowed us to select some potential biomarkers. Four of them were identified by mass spectrometry and antibody based methods. Multivariate analysis generated models that could classify samples with good sensitivity and specificity (minimum 80%) discriminating groups of patients. This analysis was used as a tool to classify peaks according to differences in level on spectra through the four categories of patients. Four biomarkers showing important diagnostic value were purified, identified (PF4, MRP8, FIBA and Hpalpha2) and two of these: PF4 and Hpalpha2 were detected in sera by classical methods. SELDI-TOF-MS technology and use of the multiple decision trees method led to protein biomarker patterns analysis and allowed the selection of potential individual biomarkers. Their downstream identification may reveal to be helpful for IBD classification and etiology understanding. PMID:17258689

  13. The human liver-specific proteome defined by transcriptomics and antibody-based profiling.

    PubMed

    Kampf, Caroline; Mardinoglu, Adil; Fagerberg, Linn; Hallström, Björn M; Edlund, Karolina; Lundberg, Emma; Pontén, Fredrik; Nielsen, Jens; Uhlen, Mathias

    2014-07-01

    Human liver physiology and the genetic etiology of the liver diseases can potentially be elucidated through the identification of proteins with enriched expression in the liver. Here, we combined data from RNA sequencing (RNA-Seq) and antibody-based immunohistochemistry across all major human tissues to explore the human liver proteome with enriched expression, as well as the cell type-enriched expression in hepatocyte and bile duct cells. We identified in total 477 protein-coding genes with elevated expression in the liver: 179 genes have higher expression as compared to all the other analyzed tissues; 164 genes have elevated transcript levels in the liver shared with at least one other tissue type; and an additional 134 genes have a mild level of increased expression in the liver. We identified the precise localization of these proteins through antibody-based protein profiling and the subcellular localization of these proteins through immunofluorescent-based profiling. We also identified the biological processes and metabolic functions associated with these proteins, investigated their contribution in the occurrence of liver diseases, and identified potential targets for their treatment. Our study demonstrates the use of RNA-Seq and antibody-based immunohistochemistry for characterizing the human liver proteome, as well as the use of tissue-specific proteins in identification of novel drug targets and discovery of biomarkers.-Kampf, C., Mardinoglu, A., Fagerberg, L., Hallström, B. M., Edlund, K., Lundberg, E., Pontén, F., Nielsen, J., Uhlen, M. The human liver-specific proteome defined by transcriptomics and antibody-based profiling. PMID:24648543

  14. Proteomic Profiling of Fast-To-Slow Muscle Transitions during Aging

    PubMed Central

    Ohlendieck, Kay

    2011-01-01

    Old age is associated with a large spectrum of physical ailments, including muscle wasting. Skeletal muscle degeneration drastically increases the risk of poor balance, frequent falling and impaired mobility in the elderly. In order to identify new therapeutic targets to halt or even reverse age-dependent muscle weakness and improve diagnostic methods to properly evaluate sarcopenia as a common geriatric syndrome, there is an urgent need to establish a reliable biomarker signature of muscle aging. In this respect, mass spectrometry-based proteomics has been successfully applied for studying crude extracts and subcellular fractions from aged animal and human muscle tissues to identify novel aging marker proteins. This review focuses on key physiological and metabolic aspects of sarcopenia, i.e., age-related muscle fiber transitions and metabolic shifts in aging muscle as revealed by proteomics. Over the last decade, proteomic profiling studies have clearly confirmed the idea that sarcopenia is based on a multi-factorial pathophysiology and that a glycolytic-to-oxidative shift occurs in slower-twitching senescent muscles. Both, newly identified protein factors and confirmed alterations in crucial metabolic and contractile elements can now be employed to establish a sarcopenia-specific biomarker signature. PMID:22207852

  15. S-Nitrosylation Proteome Profile of Peripheral Blood Mononuclear Cells in Human Heart Failure

    PubMed Central

    Spratt, Heidi M.; Gupta, Shivali; Petersen, John R.; Kuyumcu-Martinez, Muge N.

    2016-01-01

    Nitric oxide (NO) protects the heart against ischemic injury; however, NO- and superoxide-dependent S-nitrosylation (S-NO) of cysteines can affect function of target proteins and play a role in disease outcome. We employed 2D-GE with thiol-labeling FL-maleimide dye and MALDI-TOF MS/MS to capture the quantitative changes in abundance and S-NO proteome of HF patients (versus healthy controls, n = 30/group). We identified 93 differentially abundant (59-increased/34-decreased) and 111 S-NO-modified (63-increased/48-decreased) protein spots, respectively, in HF subjects (versus controls, fold-change | ≥1.5|, p ≤ 0.05). Ingenuity pathway analysis of proteome datasets suggested that the pathways involved in phagocytes' migration, free radical production, and cell death were activated and fatty acid metabolism was decreased in HF subjects. Multivariate adaptive regression splines modeling of datasets identified a panel of proteins that will provide >90% prediction success in classifying HF subjects. Proteomic profiling identified ATP-synthase, thrombospondin-1 (THBS1), and vinculin (VCL) as top differentially abundant and S-NO-modified proteins, and these proteins were verified by Western blotting and ELISA in different set of HF subjects. We conclude that differential abundance and S-NO modification of proteins serve as a mechanism in regulating cell viability and free radical production, and THBS1 and VCL evaluation will potentially be useful in the prediction of heart failure.

  16. Multiplexed, Proteome-Wide Protein Expression Profiling: Yeast Deubiquitylating Enzyme Knockout Strains

    PubMed Central

    Isasa, Marta; Rose, Christopher M.; Elsasser, Suzanne; Navarrete-Perea, José; Paulo, Joao A.; Finley, Daniel J.; Gygi, Steven P.

    2016-01-01

    Characterizing a protein’s function often requires a description of the cellular state in its absence. Multiplexing in mass spectrometry-based proteomics has now achieved the ability to globally measure protein expression levels in yeast from 10 cell states simultaneously. We applied this approach to quantify expression differences in wild type and nine deubiquitylating enzyme (DUB) knockout strains with the goal of creating “information networks” that might provide deeper, mechanistic insights into a protein’s biological role. In total, more than 3700 proteins were quantified with high reproducibility across three biological replicates (30 samples in all). DUB mutants demonstrated different proteomics profiles, consistent with distinct roles for each family member. These included differences in total ubiquitin levels and specific chain linkages. Moreover, specific expression changes suggested novel functions for several DUB family members. For instance, the ubp3Δ mutant showed large expression changes for members of the cytochrome C oxidase complex, consistent with a role for Ubp3 in mitochondrial regulation. Several DUBs also showed broad expression changes for phosphate transporters as well as other components of the inorganic phosphate signaling pathway, suggesting a role for these DUBs in regulating phosphate metabolism. These data highlight the potential of multiplexed proteome-wide analyses for biological investigation and provide a framework for further study of the DUB family. Our methods are readily applicable to the entire collection of yeast deletion mutants and may help facilitate systematic analysis of yeast and other organisms. PMID:26503604

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

    PubMed

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

    2015-06-01

    Nonfunctional pituitary adenoma (NFPA) is highly heterogeneous with different hormone-expressed subtypes in NFPA tissues including follicle-stimulating hormone (FSH) positive, luteinizing hormone-positive, FSH/luteinizing hormone-positive, and negative types. To analyze in-depth the variations in the proteomes among different NFPA subtypes for our long-term goal to clarify molecular mechanisms of NFPA and to detect tumor biomarker for personalized medicine practice, a reference map of proteome of a human FSH-expressed NFPA tissue was described here. 2DE and PDQuest image analysis were used to array each protein. MALDI-TOF PMF and human Swiss-Prot databases with MASCOT search were used to identify each protein. A good 2DE pattern with high level of between-gel reproducibility was attained with an average positional deviation 1.98 ± 0.75 mm in the IEF direction and 1.62 ± 0.68 mm in the SDS-PAGE direction. Approximately 1200 protein spots were 2DE-detected and 192 redundant proteins that were contained in 141 protein spots were PMF-identified, representing 107 nonredundant proteins. Those proteins were located in cytoplasm, nucleus, plasma membrane, extracellular space, and so on, and those functioned in transmembrane receptor, ion channel, transcription/translation regulator, transporter, enzyme, phosphatase, kinase, and so on. Several important pathway networks were characterized from those identified proteins with DAVID and Ingenuity Pathway Analysis systems, including gluconeogenesis and glycolysis, mitochondrial dysfunction, oxidative stress, cell-cycle alteration, MAPKsignaling system, immune response, TP53-signaling, VEGF-signaling, and inflammation signaling pathways. Those resulting data contribute to a functional profile of the proteome of a human FSH-positive NFPA tissue, and will serve as a reference for the heterogeneity analysis of NFPA proteomes. PMID:25809007

  18. Dynamic proteomic profiling of a unicellular cyanobacterium Cyanothece ATCC51142 across light-dark diurnal cycles

    PubMed Central

    2011-01-01

    Background Unicellular cyanobacteria of the genus Cyanothece are recognized for their ability to execute nitrogen (N2)-fixation in the dark and photosynthesis in the light. An understanding of these mechanistic processes in an integrated systems context should provide insights into how Cyanothece might be optimized for specialized environments and/or industrial purposes. Systems-wide dynamic proteomic profiling with mass spectrometry (MS) analysis should reveal fundamental insights into the control and regulation of these functions. Results To expand upon the current knowledge of protein expression patterns in Cyanothece ATCC51142, we performed quantitative proteomic analysis using partial ("unsaturated") metabolic labeling and high mass accuracy LC-MS analysis. This dynamic proteomic profiling identified 721 actively synthesized proteins with significant temporal changes in expression throughout the light-dark cycles, of which 425 proteins matched with previously characterized cycling transcripts. The remaining 296 proteins contained a cluster of proteins uniquely involved in DNA replication and repair, protein degradation, tRNA synthesis and modification, transport and binding, and regulatory functions. Functional classification of labeled proteins suggested that proteins involved in respiration and glycogen metabolism showed increased expression in the dark cycle together with nitrogenase, suggesting that N2-fixation is mediated by higher respiration and glycogen metabolism. Results indicated that Cyanothece ATCC51142 might utilize alternative pathways for carbon (C) and nitrogen (N) acquisition, particularly, aspartic acid and glutamate as substrates of C and N, respectively. Utilization of phosphoketolase (PHK) pathway for the conversion of xylulose-5P to pyruvate and acetyl-P likely constitutes an alternative strategy to compensate higher ATP and NADPH demand. Conclusion This study provides a deeper systems level insight into how Cyanothece ATCC51142

  19. A Miniaturized Chemical Proteomic Approach for Target Profiling of Clinical Kinase Inhibitors in Tumor Biopsies

    PubMed Central

    Chamrád, Ivo; Rix, Uwe; Stukalov, Alexey; Gridling, Manuela; Parapatics, Katja; Müller, André C.; Altiok, Soner; Colinge, Jacques; Superti-Furga, Giulio; Haura, Eric B.; Bennett, Keiryn L.

    2014-01-01

    While targeted therapy based on the idea of attenuating the activity of a preselected, therapeutically relevant protein has become one of the major trends in modern cancer therapy, no truly specific targeted drug has been developed and most clinical agents have displayed a degree of polypharmacology. Therefore, the specificity of anticancer therapeutics has emerged as a highly important but severely underestimated issue. Chemical proteomics is a powerful technique combining postgenomic drug-affinity chromatography with high-end mass spectrometry analysis and bioinformatic data processing to assemble a target profile of a desired therapeutic molecule. Due to high demands on the starting material, however, chemical proteomic studies have been mostly limited to cancer cell lines. Herein, we report a down-scaling of the technique to enable the analysis of very low abundance samples, as those obtained from needle biopsies. By a systematic investigation of several important parameters in pull-downs with the multikinase inhibitor bosutinib, the standard experimental protocol was optimized to 100 µg protein input. At this level, more than 30 well-known targets were detected per single pull-down replicate with high reproducibility. Moreover, as presented by the comprehensive target profile obtained from miniaturized pull-downs with another clinical drug, dasatinib, the optimized protocol seems to be extendable to other drugs of interest. Sixty distinct human and murine targets were finally identified for bosutinib and dasatinib in chemical proteomic experiments utilizing core needle biopsy samples from xenotransplants derived from patient tumor tissue. Altogether, the developed methodology proves robust and generic and holds many promises for the field of personalized health care. PMID:23901793

  20. Comparative proteomic analysis of four Bacillus clausii strains: proteomic expression signature distinguishes protein profile of the strains.

    PubMed

    Lippolis, Rosa; Gnoni, Antonio; Abbrescia, Anna; Panelli, Damiano; Maiorano, Stefania; Paternoster, Maria Stefania; Sardanelli, Anna Maria; Papa, Sergio; Gaballo, Antonio

    2011-11-18

    A comparative proteomic approach, using two dimensional gel electrophoresis and mass spectrometry, has been developed to compare and elucidate the differences among the cellular proteomes of four closely related isogenic O/C, SIN, N/R and T, B. clausii strains during both exponential and stationary phases of growth. Image analysis of the electropherograms reveals a high degree of concordance among the four proteomes, some proteins result, however, differently expressed. The proteins spots exhibiting high different expression level were identified, by mass-spectrometry analysis, as alcohol dehydrogenase (ADHA, EC1.2.1.3; ABC0046 isoform) aldehyde dehydrogenase (DHAS, EC 1.2.1.3; ABC0047 isoform) and flagellin-protein of B. clausii KSM-k16. The different expression levels of the two dehydrogenases were confirmed by quantitative RT-PCR and dehydrogenases enzymatic activity. The different patterns of protein expression can be considered as cell proteome signatures of the different strains. PMID:21810490

  1. Lung Cancer Signatures in Plasma Based on Proteome Profiling of Mouse Tumor Models

    PubMed Central

    Taguchi, Ayumu; Politi, Katerina; Pitteri, Sharon J.; Lockwood, William W.; Faça, Vitor M.; Kelly-Spratt, Karen; Wong, Chee-Hong; Zhang, Qing; Chin, Alice; Park, Kwon-Sik; Goodman, Gary; Gazdar, Adi F.; Sage, Julien; Dinulescu, Daniela M.; Kucherlapati, Raju; DePinho, Ronald A.; Kemp, Christopher J.; Varmus, Harold E.; Hanash, Samir M.

    2012-01-01

    SUMMARY We investigated the potential of in-depth quantitative proteomics to reveal plasma protein signatures that reflect lung tumor biology. We compared plasma protein profiles of four mouse models of lung cancer with profiles of models of pancreatic, ovarian, colon, prostate, and breast cancer and two models of inflammation. A protein signature for Titf1/Nkx2-1, a known lineage-survival oncogene in lung cancer, was found in plasmas of mouse models of lung adenocarcinoma. An EGFR signature was found in plasma of an EGFR mutant model, and a distinct plasma signature related to neuroendocrine development was uncovered in the small-cell lung cancer model. We demonstrate relevance to human lung cancer of the protein signatures identified on the basis of mouse models. PMID:21907921

  2. Lung cancer signatures in plasma based on proteome profiling of mouse tumor models.

    PubMed

    Taguchi, Ayumu; Politi, Katerina; Pitteri, Sharon J; Lockwood, William W; Faça, Vitor M; Kelly-Spratt, Karen; Wong, Chee-Hong; Zhang, Qing; Chin, Alice; Park, Kwon-Sik; Goodman, Gary; Gazdar, Adi F; Sage, Julien; Dinulescu, Daniela M; Kucherlapati, Raju; Depinho, Ronald A; Kemp, Christopher J; Varmus, Harold E; Hanash, Samir M

    2011-09-13

    We investigated the potential of in-depth quantitative proteomics to reveal plasma protein signatures that reflect lung tumor biology. We compared plasma protein profiles of four mouse models of lung cancer with profiles of models of pancreatic, ovarian, colon, prostate, and breast cancer and two models of inflammation. A protein signature for Titf1/Nkx2-1, a known lineage-survival oncogene in lung cancer, was found in plasmas of mouse models of lung adenocarcinoma. An EGFR signature was found in plasma of an EGFR mutant model, and a distinct plasma signature related to neuroendocrine development was uncovered in the small-cell lung cancer model. We demonstrate relevance to human lung cancer of the protein signatures identified on the basis of mouse models. PMID:21907921

  3. Age and gender specific biokinetic model for strontium in humans.

    PubMed

    Shagina, N B; Tolstykh, E I; Degteva, M O; Anspaugh, L R; Napier, B A

    2015-03-01

    A biokinetic model for strontium in humans is necessary for quantification of internal doses due to strontium radioisotopes. The ICRP-recommended biokinetic model for strontium has limitations for use in a population study, because it is not gender specific and does not cover all age ranges. The extensive Techa River data set on (90)Sr in humans (tens of thousands of measurements) is a unique source of data on long-term strontium retention for men and women of all ages at intake. These, as well as published data, were used for evaluation of age- and gender-specific parameters for a new compartment biokinetic model for strontium (Sr-AGe model). The Sr-AGe model has a similar structure to the ICRP model for the alkaline earth elements. The following parameters were mainly re-evaluated: gastrointestinal absorption and parameters related to the processes of bone formation and resorption defining calcium and strontium transfers in skeletal compartments. The Sr-AGe model satisfactorily describes available data sets on strontium retention for different kinds of intake (dietary and intravenous) at different ages (0-80 years old) and demonstrates good agreement with data sets for different ethnic groups. The Sr-AGe model can be used for dose assessment in epidemiological studies of general populations exposed to ingested strontium radioisotopes. PMID:25574605

  4. Age and gender specific biokinetic model for strontium in humans

    SciTech Connect

    Shagina, N. B.; Tolstykh, E. I.; Degteva, M. O.; Anspaugh, L. R.; Napier, Bruce A.

    2015-03-01

    A biokinetic model for strontium in humans is necessary for quantification of internal doses due to strontium radioisotopes. The ICRP-recommended biokinetic model for strontium has limitation for use in a population study, because it is not gender specific and does not cover all age ranges. The extensive Techa River data set on 90Sr in humans (tens of thousands of measurements) is a unique source of data on long-term strontium retention for men and women of all ages at intake. These, as well as published data, were used for evaluation of age- and gender-specific parameters for a new compartment biokinetic model for strontium (Sr-AGe model). The Sr-AGe model has similar structure as the ICRP model for the alkaline earth elements. The following parameters were mainly reevaluated: gastro-intestinal absorption and parameters related to the processes of bone formation and resorption defining calcium and strontium transfers in skeletal compartments. The Sr-AGe model satisfactorily describes available data sets on strontium retention for different kinds of intake (dietary and intravenous) at different ages (0–80 years old) and demonstrates good agreement with data sets for different ethnic groups. The Sr-AGe model can be used for dose assessment in epidemiological studies of general population exposed to ingested strontium radioisotopes.

  5. Proteome profiling of human neutrophil granule subsets, secretory vesicles, and cell membrane: correlation with transcriptome profiling of neutrophil precursors.

    PubMed

    Rørvig, Sara; Østergaard, Ole; Heegaard, Niels H H; Borregaard, Niels

    2013-10-01

    Neutrophils are indispensable in the innate immune defense against invading microorganisms. Neutrophils contain SVs and several subsets of granules that are essential for their function. Proteins present in neutrophil SVs and granules are synthesized during terminal granulopoiesis in the bone marrow. The heterogeneity of granules, as determined by marker proteins characteristic of each granule subset, is thought to result from differences in the biosynthetic windows of major classes of granule proteins, a process referred to as targeting by timing. Qualitative proteomic analysis of neutrophil granules, SVs, and plasma membrane has been performed before. Here, we performed subcellular fractionation on freshly isolated human neutrophils by nitrogen cavitation and density centrifugation on a four-layer Percoll gradient. Granule subsets were pooled and subjected to SDS-PAGE, and gel pieces were in-gel-digested with trypsin. The resulting peptides were analyzed using LTQ Orbitrap XL tandem MS. A total of 1292 unique proteins were identified and grouped, according to the neutrophil fraction, in which they displayed maximal expression. In addition to various known neutrophil proteins, several uncharacterized proteins were found, as well as proteins not described previously in neutrophils. To study the correlation between mRNA expression in neutrophil precursors and the localization of their cognate proteins, the distribution of 126 identified proteins was compared with their mRNA expression profiles. The neutrophil subcellular proteome profiles presented here may be used as a database in combination with the mRNA array database to predict and test the presence and localization of proteins in neutrophil granules and membranes. PMID:23650620

  6. Proteome Profile of Swine Testicular Cells Infected with Porcine Transmissible Gastroenteritis Coronavirus

    PubMed Central

    Ma, Ruili; Zhang, Yanming; Liu, Haiquan; Ning, Pengbo

    2014-01-01

    The interactions occurring between a virus and a host cell during a viral infection are complex. The purpose of this paper was to analyze altered cellular protein levels in porcine transmissible gastroenteritis coronavirus (TGEV)-infected swine testicular (ST) cells in order to determine potential virus-host interactions. A proteomic approach using isobaric tags for relative and absolute quantitation (iTRAQ)-coupled two-dimensional liquid chromatography-tandem mass spectrometry identification was conducted on the TGEV-infected ST cells. The results showed that the 4-plex iTRAQ-based quantitative approach identified 4,112 proteins, 146 of which showed significant changes in expression 48 h after infection. At 64 h post infection, 219 of these proteins showed significant change, further indicating that a larger number of proteomic changes appear to occur during the later stages of infection. Gene ontology analysis of the altered proteins showed enrichment in multiple biological processes, including cell adhesion, response to stress, generation of precursor metabolites and energy, cell motility, protein complex assembly, growth, developmental maturation, immune system process, extracellular matrix organization, locomotion, cell-cell signaling, neurological system process, and cell junction organization. Changes in the expression levels of transforming growth factor beta 1 (TGF-β1), caspase-8, and heat shock protein 90 alpha (HSP90α) were also verified by western blot analysis. To our knowledge, this study is the first time the response profile of ST host cells following TGEV infection has been analyzed using iTRAQ technology, and our description of the late proteomic changes that are occurring after the time of vigorous viral production are novel. Therefore, this study provides a solid foundation for further investigation, and will likely help us to better understand the mechanisms of TGEV infection and pathogenesis. PMID:25333634

  7. Proteomic Profiling and Protein Identification by MALDI-TOF Mass Spectrometry in Unsequenced Parasitic Nematodes

    PubMed Central

    Millares, Paul; LaCourse, E. James; Perally, Samirah; Ward, Deborah A.; Prescott, Mark C.; Hodgkinson, Jane E.; Brophy, Peter M.; Rees, Huw H.

    2012-01-01

    Lack of genomic sequence data and the relatively high cost of tandem mass spectrometry have hampered proteomic investigations into helminths, such as resolving the mechanism underpinning globally reported anthelmintic resistance. Whilst detailed mechanisms of resistance remain unknown for the majority of drug-parasite interactions, gene mutations and changes in gene and protein expression are proposed key aspects of resistance. Comparative proteomic analysis of drug-resistant and -susceptible nematodes may reveal protein profiles reflecting drug-related phenotypes. Using the gastro-intestinal nematode, Haemonchus contortus as case study, we report the application of freely available expressed sequence tag (EST) datasets to support proteomic studies in unsequenced nematodes. EST datasets were translated to theoretical protein sequences to generate a searchable database. In conjunction with matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), Peptide Mass Fingerprint (PMF) searching of databases enabled a cost-effective protein identification strategy. The effectiveness of this approach was verified in comparison with MS/MS de novo sequencing with searching of the same EST protein database and subsequent searches of the NCBInr protein database using the Basic Local Alignment Search Tool (BLAST) to provide protein annotation. Of 100 proteins from 2-DE gel spots, 62 were identified by MALDI-TOF-MS and PMF searching of the EST database. Twenty randomly selected spots were analysed by electrospray MS/MS and MASCOT Ion Searches of the same database. The resulting sequences were subjected to BLAST searches of the NCBI protein database to provide annotation of the proteins and confirm concordance in protein identity from both approaches. Further confirmation of protein identifications from the MS/MS data were obtained by de novo sequencing of peptides, followed by FASTS algorithm searches of the EST putative protein database. This

  8. Urinary proteomic profiling reveals diclofenac-induced renal injury and hepatic regeneration in mice

    SciTech Connect

    Swelm, Rachel P.L. van; Laarakkers, Coby M.M.; Pertijs, Jeanne C.L.M.; Verweij, Vivienne; Masereeuw, Rosalinde; Russel, Frans G.M.

    2013-06-01

    Diclofenac (DF) is a widely used non-steroidal anti-inflammatory drug for the treatment of rheumatic disorders, but is often associated with liver injury. We applied urinary proteomic profiling using MALDI-TOF MS to identify biomarkers for DF-induced hepatotoxicity in mice. Female CH3/HeOUJIco mice were treated with 75 mg/kg bw DF by oral gavage and 24 h urine was collected. Proteins identified in urine of DF-treated mice included epidermal growth factor, transthyretin, kallikrein, clusterin, fatty acid binding protein 1 and urokinase, which are related to liver regeneration but also to kidney injury. Both organs showed enhanced levels of oxidative stress (TBARS, p < 0.01). Kidney injury was confirmed by histology and increased Kim1 and Il-6 mRNA expression levels (p < 0.001 and p < 0.01). Liver histology and plasma ALT levels in DF-treated mice were not different from control, but mRNA expression of Stat3 (p < 0.001) and protein expression of PCNA (p < 0.05) were increased, indicating liver regeneration. In conclusion, urinary proteome analysis revealed that DF treatment in mice induced kidney and liver injury. Within 24 h, however, the liver was able to recover by activating tissue regeneration processes. Hence, the proteins found in urine of DF-treated mice represent kidney damage rather than hepatic injury. - Highlights: • The urinary proteome shows biological processes involved in adverse drug reactions. • Urine proteins of DF-treated mice relate to kidney injury rather than liver injury. • Liver regeneration, not liver injury, is apparent 24h after oral DF administration. • Pretreatment with LPS does not enhance DF-induced liver injury in mice.

  9. Changes in the proteomic profile of adipose tissue-derived mesenchymal stem cells during passages

    PubMed Central

    2012-01-01

    Background Human mesenchymal stem cells (hMSC) have recently raised the attention because of their therapeutic potential in the novel context of regenerative medicine. However, the safety of these new and promising cellular products should be carefully defined before they can be used in the clinical setting, as. The protein expression profile of these cells might reveal potential hazards associated with senescence and tumoral transformation which may occur during culture. Proteomic is a valuable tool for hMSC characterization and identification of possible changes during expansion. Results We used Surface Enhanced Laser Desorption/Ionization-Time Of Flight-Mass Spectrometry (SELDI-ToF-MS) to evaluate the presence of stable molecular markers in adipose tissue-derived mesenchymal stem cells (AD-MSC) produced under conditions of good manufacturing practices (GMP). Proteomic patterns of cells prepared were consistent, with 4 up-regulated peaks (mass-to-charge ratio (m/z) 8950, 10087, 10345, and 13058) through subculture steps (P0-P7) with similar trend in three donors. Among the differentially expressed proteins found in the cytoplasmic and nuclear fractions, a cytoplasmic 10.1 kDa protein was upregulated during culture passages and was identified as S100A6 (Calcyclin). Conclusions This study suggests for the first time that common variation could occur in AD-MSC from different donors, with the identification of S100A6, a protein prevalently related to cell proliferation and cell culture condition. These results support the hypothesis of common proteomic changes during MSCs expansion and could give important insight in the knowledge of molecular mechanisms intervening during MSC expansion. PMID:22828447

  10. Proteomic Profiling in the Brain of CLN1 Disease Model Reveals Affected Functional Modules.

    PubMed

    Tikka, Saara; Monogioudi, Evanthia; Gotsopoulos, Athanasios; Soliymani, Rabah; Pezzini, Francesco; Scifo, Enzo; Uusi-Rauva, Kristiina; Tyynelä, Jaana; Baumann, Marc; Jalanko, Anu; Simonati, Alessandro; Lalowski, Maciej

    2016-03-01

    Neuronal ceroid lipofuscinoses (NCL) are the most commonly inherited progressive encephalopathies of childhood. Pathologically, they are characterized by endolysosomal storage with different ultrastructural features and biochemical compositions. The molecular mechanisms causing progressive neurodegeneration and common molecular pathways linking expression of different NCL genes are largely unknown. We analyzed proteome alterations in the brains of a mouse model of human infantile CLN1 disease-palmitoyl-protein thioesterase 1 (Ppt1) gene knockout and its wild-type age-matched counterpart at different stages: pre-symptomatic, symptomatic and advanced. For this purpose, we utilized a combination of laser capture microdissection-based quantitative liquid chromatography tandem mass spectrometry (MS) and matrix-assisted laser desorption/ionization time-of-flight MS imaging to quantify/visualize the changes in protein expression in disease-affected brain thalamus and cerebral cortex tissue slices, respectively. Proteomic profiling of the pre-symptomatic stage thalamus revealed alterations mostly in metabolic processes and inhibition of various neuronal functions, i.e., neuritogenesis. Down-regulation in dynamics associated with growth of plasma projections and cellular protrusions was further corroborated by findings from RNA sequencing of CLN1 patients' fibroblasts. Changes detected at the symptomatic stage included: mitochondrial functions, synaptic vesicle transport, myelin proteome and signaling cascades, such as RhoA signaling. Considerable dysregulation of processes related to mitochondrial cell death, RhoA/Huntington's disease signaling and myelin sheath breakdown were observed at the advanced stage of the disease. The identified changes in protein levels were further substantiated by bioinformatics and network approaches, immunohistochemistry on brain tissues and literature knowledge, thus identifying various functional modules affected in the CLN1 childhood

  11. Changes of human serum proteome profile during 7-day “dry” immersion

    NASA Astrophysics Data System (ADS)

    Pakharukova, N. A.; Pastushkova, L. Kh.; Larina, I. M.; Grigoriev, A. I.

    2011-05-01

    The aim of this study was to characterize changes of serum proteome profile during 7-day "dry" immersion (DI). The experiment with DI consisted of three series: control group without countermeasures (10 men), with using mechanical stimulation (6 men) and low-frequency myostimulation (5 men) as preventive means. Serum samples were fractionated using ClinProt robot (Bruker Daltonics) on magnetic beads (weak cation exchange magnetic beads—MB WCX) prior to mass-spectral profiling. It was obtained 170 peaks after fractionation of serum samples in each group. On 7th immersion day peak areas of fibrinopeptide A ( m/ z=1206; 1464), angiotensin II ( m/ z=1051), high molecular mass kininogen fragment ( m/ z=2133 Da) and C3-fragment of the complement system ( m/ z=1350 Da) were significantly decreased comparing with pre-experimental values of all experimental series. Peak areas of apolipoprotein C III ( m/ z=9419) and C4a fragment of the complement system ( m/ z=3206 Da) were increased. On 7th day of the recovery peak areas of all changed peaks were not close to pre-experimental values. This fact provided evidence of incomplete recovery of an organism after DI. The depth of the alterations had considerable individual variability. Thereby the detected changes of serum proteome profile in the experiment. They indicated a reorganization of the hormonal, immune systems and lipid metabolism. The use of myostimulation and mechanical stimulation as countermeasures partly compensated adverse effects of 7-day dry immersion on the parameters of coagulation system (fibrinopeptide A) and lipid metabolism (apolipoprotein CIII).

  12. Analysis of biostimulated microbial communities from two field experiments reveals temporal and spatial differences in proteome profiles

    SciTech Connect

    Callister, S.J.; Wilkins, M.J.; Nicora, C.D.; Williams, K.H.; Banfield, J.F.; VerBerkmoes, N.C.; Hettich, R.L.; NGuessan, A.L.; Mouser, P.J.; Elifantz, H.; Smith, R.D.; Lovley, D.R.; Lipton, M.S.; Long, P.E.

    2010-07-15

    Stimulated by an acetate-amendment field experiment conducted in 2007, anaerobic microbial populations in the aquifer at the Rifle Integrated Field Research Challenge site in Colorado reduced mobile U(VI) to insoluble U(IV). During this experiment, planktonic biomass was sampled at various time points to quantitatively evaluate proteomes. In 2008, an acetate-amended field experiment was again conducted in a similar manner to the 2007 experiment. As there was no comprehensive metagenome sequence available for use in proteomics analysis, we systematically evaluated 12 different organism genome sequences to generate sets of aggregate genomes, or “pseudo-metagenomes”, for supplying relative quantitative peptide and protein identifications. Proteomics results support previous observations of the dominance of Geobacteraceae during biostimulation using acetate as sole electron donor, and revealed a shift from an early stage of iron reduction to a late stage of iron reduction. Additionally, a shift from iron reduction to sulfate reduction was indicated by changes in the contribution of proteome information contributed by different organism genome sequences within the aggregate set. In addition, the comparison of proteome measurements made between the 2007 field experiment and 2008 field experiment revealed differences in proteome profiles. These differences may be the result of alterations in abundance and population structure within the planktonic biomass samples collected for analysis.

  13. Analysis of Biostimulated Microbial Communities from Two Field Experiments Reveals Temporal and Spatial Differences in Proteome Profiles

    SciTech Connect

    Callister, Stephen J; Wilkins, Mike; Nicora, Carrie D.; Williams, Ken; Banfield, Jillian F.; Verberkmoes, Nathan C; Hettich, Robert {Bob} L; N'Guessan, A. Lucie; Mouser, Paula J; Elifantz, Hila; Smith, Richard D.; Lovley, Derek; Lipton, Mary S; Long, Phil

    2010-01-01

    Stimulated by an acetate-amendment field experiment conducted in 2007, anaerobic microbial populations in the aquifer at the Rifle Integrated Field Research Challenge site in Colorado reduced mobile U(VI) to insoluble U(IV). During this experiment, planktonic biomass was sampled at various time points to quantitatively evaluate proteomes. In 2008, an acetateamended field experiment was again conducted in a similar manner to the 2007 experiment. As there was no comprehensive metagenome sequence available for use in proteomics analysis, we systematically evaluated 12 different organism genome sequences to generate sets of aggregate genomes, or pseudo-metagenomes , for supplying relative quantitative peptide and protein identifications. Proteomics results support previous observations of the dominance of Geobacteraceae during biostimulation using acetate as sole electron donor, and revealed a shift from an early stage of iron reduction to a late stage of iron reduction. Additionally,ashift from iron reduction to sulfate reduction was indicated by changes in the contribution of proteome information contributed by different organism genome sequences within the aggregate set. In addition, the comparison of proteome measurements made between the 2007 field experiment and 2008 field experiment revealed differences in proteome profiles. These differences may be the result of alterations in abundance and population structure within the planktonic biomass samples collected for analysis.

  14. Human hair shaft proteomic profiling: individual differences, site specificity and cuticle analysis.

    PubMed

    Laatsch, Chelsea N; Durbin-Johnson, Blythe P; Rocke, David M; Mukwana, Sophie; Newland, Abby B; Flagler, Michael J; Davis, Michael G; Eigenheer, Richard A; Phinney, Brett S; Rice, Robert H

    2014-01-01

    Hair from different individuals can be distinguished by physical properties. Although some data exist on other species, examination of the individual molecular differences within the human hair shaft has not been thoroughly investigated. Shotgun proteomic analysis revealed considerable variation in profile among samples from Caucasian, African-American, Kenyan and Korean subjects. Within these ethnic groups, prominent keratin proteins served to distinguish individual profiles. Differences between ethnic groups, less marked, relied to a large extent on levels of keratin associated proteins. In samples from Caucasian subjects, hair shafts from axillary, beard, pubic and scalp regions exhibited distinguishable profiles, with the last being most different from the others. Finally, the profile of isolated hair cuticle cells was distinguished from that of total hair shaft by levels of more than 20 proteins, the majority of which were prominent keratins. The cuticle also exhibited relatively high levels of epidermal transglutaminase (TGM3), accounting for its observed low degree of protein extraction by denaturants. In addition to providing insight into hair structure, present findings may lead to improvements in differentiating hair from various ethnic origins and offer an approach to extending use of hair in crime scene evidence for distinguishing among individuals. PMID:25165623

  15. Streptococcus iniae SF1: Complete Genome Sequence, Proteomic Profile, and Immunoprotective Antigens

    PubMed Central

    Zhang, Bao-cun; Zhang, Jian; Sun, Li

    2014-01-01

    Streptococcus iniae is a Gram-positive bacterium that is reckoned one of the most severe aquaculture pathogens. It has a broad host range among farmed marine and freshwater fish and can also cause zoonotic infection in humans. Here we report for the first time the complete genome sequence as well as the host factor-induced proteomic profile of a pathogenic S. iniae strain, SF1, a serotype I isolate from diseased fish. SF1 possesses a single chromosome of 2,149,844 base pairs, which contains 2,125 predicted protein coding sequences (CDS), 12 rRNA genes, and 45 tRNA genes. Among the protein-encoding CDS are genes involved in resource acquisition and utilization, signal sensing and transduction, carbohydrate metabolism, and defense against host immune response. Potential virulence genes include those encoding adhesins, autolysins, toxins, exoenzymes, and proteases. In addition, two putative prophages and a CRISPR-Cas system were found in the genome, the latter containing a CRISPR locus and four cas genes. Proteomic analysis detected 21 secreted proteins whose expressions were induced by host serum. Five of the serum-responsive proteins were subjected to immunoprotective analysis, which revealed that two of the proteins were highly protective against lethal S. iniae challenge when used as purified recombinant subunit vaccines. Taken together, these results provide an important molecular basis for future study of S. iniae in various aspects, in particular those related to pathogenesis and disease control. PMID:24621602

  16. The advantage of laser-capture microdissection over whole tissue analysis in proteomic profiling studies.

    PubMed

    De Marchi, Tommaso; Braakman, Rene B H; Stingl, Christoph; van Duijn, Martijn M; Smid, Marcel; Foekens, John A; Luider, Theo M; Martens, John W M; Umar, Arzu

    2016-05-01

    Laser-capture microdissection (LCM) offers a reliable cell population enrichment tool and has been successfully coupled to MS analysis. Despite this, most proteomic studies employ whole tissue lysate (WTL) analysis in the discovery of disease biomarkers and in profiling analyses. Furthermore, the influence of tissue heterogeneity in WTL analysis, nor its impact in biomarker discovery studies have been completely elucidated. In order to address this, we compared previously obtained high resolution MS data from a cohort of 38 breast cancer tissues, of which both LCM enriched tumor epithelial cells and WTL samples were analyzed. Label-free quantification (LFQ) analysis through MaxQuant software showed a significantly higher number of identified and quantified proteins in LCM enriched samples (3404) compared to WTLs (2837). Furthermore, WTL samples displayed a higher amount of missing data compared to LCM both at peptide and protein levels (p-value < 0.001). 2D analysis on co-expressed proteins revealed discrepant expression of immune system and lipid metabolisms related proteins between LCM and WTL samples. We hereby show that LCM better dissected the biology of breast tumor epithelial cells, possibly due to lower interference from surrounding tissues and highly abundant proteins. All data have been deposited in the ProteomeXchange with the dataset identifier PXD002381 (http://proteomecentral.proteomexchange.org/dataset/PXD002381). PMID:27030549

  17. Discovery of lung cancer biomarkers by profiling the plasma proteome with monoclonal antibody libraries.

    PubMed

    Guergova-Kuras, Mariana; Kurucz, István; Hempel, William; Tardieu, Nadège; Kádas, János; Malderez-Bloes, Carole; Jullien, Anne; Kieffer, Yann; Hincapie, Marina; Guttman, András; Csánky, Eszter; Dezso, Balázs; Karger, Barry L; Takács, László

    2011-12-01

    A challenge in the treatment of lung cancer is the lack of early diagnostics. Here, we describe the application of monoclonal antibody proteomics for discovery of a panel of biomarkers for early detection (stage I) of non-small cell lung cancer (NSCLC). We produced large monoclonal antibody libraries directed against the natural form of protein antigens present in the plasma of NSCLC patients. Plasma biomarkers associated with the presence of lung cancer were detected via high throughput ELISA. Differential profiling of plasma proteomes of four clinical cohorts, totaling 301 patients with lung cancer and 235 healthy controls, identified 13 lung cancer-associated (p < 0.05) monoclonal antibodies. The monoclonal antibodies recognize five different cognate proteins identified using immunoprecipitation followed by mass spectrometry. Four of the five antigens were present in non-small cell lung cancer cells in situ. The approach is capable of generating independent antibodies against different epitopes of the same proteins, allowing fast translation to multiplexed sandwich assays. Based on these results, we have verified in two independent clinical collections a panel of five biomarkers for classifying patient disease status with a diagnostics performance of 77% sensitivity and 87% specificity. Combining CYFRA, an established cancer marker, with the panel resulted in a performance of 83% sensitivity at 95% specificity for stage I NSCLC. PMID:21947365

  18. Profiling the erythrocyte membrane proteome isolated from patients diagnosed with chronic obstructive pulmonary disease.

    PubMed

    Alexandre, Bruno M; Charro, Nuno; Blonder, Josip; Lopes, Carlos; Azevedo, Pilar; Bugalho de Almeida, António; Chan, King C; Prieto, DaRue A; Issaq, Haleem; Veenstra, Timothy D; Penque, Deborah

    2012-12-01

    Structural and metabolic alterations in erythrocytes play an important role in the pathophysiology of Chronic Obstructive Pulmonary Disease (COPD). Whether these dysfunctions are related to the modulation of erythrocyte membrane proteins in patients diagnosed with COPD remains to be determined. Herein, a comparative proteomic profiling of the erythrocyte membrane fraction isolated from peripheral blood of smokers diagnosed with COPD and smokers with no COPD was performed using differential (16)O/(18)O stable isotope labeling. A total of 219 proteins were quantified as being significantly differentially expressed within the erythrocyte membrane proteomes of smokers with COPD and healthy smokers. Functional pathway analysis showed that the most enriched biofunctions were related to cell-to-cell signaling and interaction, hematological system development, immune response, oxidative stress and cytoskeleton. Chorein (VPS13A), a cytoskeleton related protein whose defects had been associated with the presence of cell membrane deformation of circulating erythrocytes was found to be down-regulated in the membrane fraction of erythrocytes obtained from COPD patients. Methemoglobin reductase (CYB5R3) was also found to be underexpressed in these cells, suggesting that COPD patients may be at higher risk for developing methemoglobinemia. This article is part of a Special Issue entitled: Integrated omics. PMID:22538302

  19. Proteomic profiling in MPTP monkey model for early Parkinson disease biomarker discovery.

    PubMed

    Lin, Xiangmin; Shi, Min; Masilamoni, Jeyaraj Gunasingh; Dator, Romel; Movius, James; Aro, Patrick; Smith, Yoland; Zhang, Jing

    2015-07-01

    Identification of reliable and robust biomarkers is crucial to enable early diagnosis of Parkinson disease (PD) and monitoring disease progression. While imperfect, the slow, chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced non-human primate animal model system of parkinsonism is an abundant source of pre-motor or early stage PD biomarker discovery. Here, we present a study of a MPTP rhesus monkey model of PD that utilizes complementary quantitative iTRAQ-based proteomic, glycoproteomics and phosphoproteomics approaches. We compared the glycoprotein, non-glycoprotein, and phosphoprotein profiles in the putamen of asymptomatic and symptomatic MPTP-treated monkeys as well as saline injected controls. We identified 86 glycoproteins, 163 non-glycoproteins, and 71 phosphoproteins differentially expressed in the MPTP-treated groups. Functional analysis of the data sets inferred the biological processes and pathways that link to neurodegeneration in PD and related disorders. Several potential biomarkers identified in this study have already been translated for their usefulness in PD diagnosis in human subjects and further validation investigations are currently under way. In addition to providing potential early PD biomarkers, this comprehensive quantitative proteomic study may also shed insights regarding the mechanisms underlying early PD development. This article is part of a Special Issue entitled: Neuroproteomics: Applications in neuroscience and neurology. PMID:25617661

  20. Immunostimulatory potential and proteome profiling of Leishmania donovani soluble exogenous antigens.

    PubMed

    Kumar, A; Samant, M; Misra, P; Khare, P; Sundar, S; Garg, Ravendra; Dube, A

    2015-07-01

    Isolation of the soluble exogenous antigens (SEAgs), its immune response study and proteome profiling is an essential prerequisite for understanding the molecular pathogenesis of Leishmania donovani. The immunostimulatory potential of L. donovani SEAgs, purified from culture of L. donovani clinical isolate, was evaluated for their ability to induce cellular responses in treated/cured hamsters. SEAgs induced significant proliferative responses in lymphocytes (SI 5.6 ± 2.3; P < 0.01) isolated from cured hamster. In addition, significant NO production in response to SEAgs was also noticed in macrophages of hamsters, mouse and human cell lines (J774A-1 and THP1). Western blot analyses with antibodies against proteophosphoglycan (PPG; surface-expressed and secreted molecule) of L. donovani revealed that PPG molecules are also present in L. donovani SEAgs. Mass spectrometry (MS)-based proteome analysis of 12 protein bands of SEAgs through MALDI-TOF/TOF endorsed the identification of some Th1-stimulatory immunogenic proteins. These immunogenic proteins may offer increased hope for the discovery of new promising vaccine candidates against visceral leishmaniasis (VL). The overall results suggest that immunostimulatory molecules are present in the SEAgs, which may be further exploited, for developing a subunit vaccine against VL a fatal human disease. PMID:25824598

  1. Discovery of Lung Cancer Biomarkers by Profiling the Plasma Proteome with Monoclonal Antibody Libraries*

    PubMed Central

    Guergova-Kuras, Mariana; Kurucz, István; Hempel, William; Tardieu, Nadège; Kádas, János; Malderez-Bloes, Carole; Jullien, Anne; Kieffer, Yann; Hincapie, Marina; Guttman, András; Csánky, Eszter; Dezső, Balázs; Karger, Barry L.; Takács, László

    2011-01-01

    A challenge in the treatment of lung cancer is the lack of early diagnostics. Here, we describe the application of monoclonal antibody proteomics for discovery of a panel of biomarkers for early detection (stage I) of non-small cell lung cancer (NSCLC). We produced large monoclonal antibody libraries directed against the natural form of protein antigens present in the plasma of NSCLC patients. Plasma biomarkers associated with the presence of lung cancer were detected via high throughput ELISA. Differential profiling of plasma proteomes of four clinical cohorts, totaling 301 patients with lung cancer and 235 healthy controls, identified 13 lung cancer-associated (p < 0.05) monoclonal antibodies. The monoclonal antibodies recognize five different cognate proteins identified using immunoprecipitation followed by mass spectrometry. Four of the five antigens were present in non-small cell lung cancer cells in situ. The approach is capable of generating independent antibodies against different epitopes of the same proteins, allowing fast translation to multiplexed sandwich assays. Based on these results, we have verified in two independent clinical collections a panel of five biomarkers for classifying patient disease status with a diagnostics performance of 77% sensitivity and 87% specificity. Combining CYFRA, an established cancer marker, with the panel resulted in a performance of 83% sensitivity at 95% specificity for stage I NSCLC. PMID:21947365

  2. Proteomic profiling reveals a catalogue of new candidate proteins for human skin aging.

    PubMed

    Laimer, Martin; Kocher, Thomas; Chiocchetti, Andreas; Trost, Andrea; Lottspeich, Friedrich; Richter, Klaus; Hintner, Helmut; Bauer, Johann W; Onder, Kamil

    2010-10-01

    Studies of skin aging are usually performed at the genomic level by investigating differentially regulated genes identified through subtractive hybridization or microarray analyses. In contrast, relatively few studies have investigated changes in protein expression of aged skin using proteomic profiling by two-dimensional (2-D) gel electrophoresis and mass spectrometry, although this approach at the protein level is suggested to reflect more accurately the aging phenotype. We undertook such a proteomic analysis of intrinsic human skin aging by quantifying proteins extracted and fluorescently labeled from sun-protected human foreskin samples pooled from 'young' and 'old' men. In addition, we analyzed these candidate gene products by 1-D and 2-D western blotting to obtain corroborative protein expression data, and by both real-time PCR (RT-PCR) and microarray analyses to confirm expression at the mRNA level. We discovered 30 putative proteins for skin aging, including previously unrecognized, post-translationally regulated candidates such as phosphatidyl-ethanolamine binding protein (PEBP) and carbonic anhydrase 1 (CA1). PMID:20849533

  3. Proteomic profiling in MPTP monkey model for early Parkinson disease biomarker discovery

    PubMed Central

    Lin, Xiangmin; Shi, Min; Gunasingh Masilamoni, Jeyaraj; Dator, Romel; Movius, James; Aro, Patrick; Smith, Yoland; Zhang, Jing

    2015-01-01

    Identification of reliable and robust biomarkers is crucial to enable early diagnosis of Parkinson disease (PD) and monitoring disease progression. While imperfect, the slow, chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced non-human primate animal model system of parkinsonism is an abundant source of pre-motor or early stage PD biomarker discovery. Here, we present a study of a MPTP rhesus monkey model of PD that utilizes complementary quantitative iTRAQ-based proteomic, glycoproteomics and phosphoproteomics approaches. We compared the glycoprotein, non-glycoprotein, and phosphoprotein profiles in the putamen of asymptomatic and symptomatic MPTP-treated monkeys as well as saline injected controls. We identified 86 glycoproteins, 163 non-glycoproteins, and 71 phosphoproteins differentially expressed in the MPTP-treated groups. Functional analysis of the data sets inferred the biological processes and pathways that link to neurodegeneration in PD and related disorders. Several potential biomarkers identified in this study have already been translated for their usefulness in PD diagnosis in human subjects and further validation investigations are currently under way. In addition to providing potential early PD biomarkers, this comprehensive quantitative proteomic study may also shed insights regarding the mechanisms underlying early PD development. This article is part of a Special Issue entitled: Neuroproteomics: Applications in neuroscience and neurology. PMID:25617661

  4. Proteomic profiling of cerebrospinal fluid identifies biomarkers for amyotrophic lateral sclerosis

    PubMed Central

    Ranganathan, Srikanth; Williams, Eric; Ganchev, Philip; Gopalakrishnan, Vanathi; Lacomis, David; Urbinelli, Leo; Newhall, Kristyn; Cudkowicz, Merit E.; Brown, Robert H.; Bowser, Robert

    2006-01-01

    Amyotrophic lateral sclerosis (ALS) is characterized by degeneration of motor neurons. We tested the hypothesis that proteomic analysis will identify protein biomarkers that provide insight into disease pathogenesis and are diagnostically useful. To identify ALS specific biomarkers, we compared the proteomic profile of cerebrospinal fluid (CSF) from ALS and control subjects using surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF-MS). We identified 30 mass ion peaks with statistically significant (p < 0.01) differences between control and ALS subjects. Initial analysis with a rule-learning algorithm yielded biomarker panels with diagnostic predictive value as subsequently assessed using an independent set of coded test subjects. Three biomarkers were identified that are either decreased (transthyretin, cystatin C) or increased (carboxy-terminal fragment of neuroendocrine protein 7B2) in ALS CSF. We validated the SELDI-TOF-MS results for transthyretin and cystatin C by immunoblot and immunohistochemistry using commercially available antibodies. These findings identify a panel of CSF protein biomarkers for ALS. PMID:16313519

  5. 2-D DIGE proteomic profiles of three strains of Fusarium graminearum grown in agmatine or glutamic acid medium

    PubMed Central

    Serchi, Tommaso; Pasquali, Matias; Leclercq, Céline C.; Planchon, Sébastien; Hoffmann, Lucien; Renaut, Jenny

    2016-01-01

    2D DIGE proteomics data obtained from three strains belonging to Fusarium graminearum s.s. species growing in a glutamic acid or agmatine containing medium are provided. A total of 381 protein species have been identified which do differ for abundance among the two treatments and among the strains (ANOVA<0.05 and abundance ratio>±1.3). Data on the diversity of protein species profiles between the two media for each strain are made available. Shared profiles among strains are discussed in Pasquali et al. [1]. Here proteins that with diverse profile can be used to differentiate strains are highlighted. The full dataset allow to obtaining single strain proteomic profiles. PMID:26981549

  6. 2-D DIGE proteomic profiles of three strains of Fusarium graminearum grown in agmatine or glutamic acid medium.

    PubMed

    Serchi, Tommaso; Pasquali, Matias; Leclercq, Céline C; Planchon, Sébastien; Hoffmann, Lucien; Renaut, Jenny

    2016-03-01

    2D DIGE proteomics data obtained from three strains belonging to Fusarium graminearum s.s. species growing in a glutamic acid or agmatine containing medium are provided. A total of 381 protein species have been identified which do differ for abundance among the two treatments and among the strains (ANOVA<0.05 and abundance ratio>±1.3). Data on the diversity of protein species profiles between the two media for each strain are made available. Shared profiles among strains are discussed in Pasquali et al. [1]. Here proteins that with diverse profile can be used to differentiate strains are highlighted. The full dataset allow to obtaining single strain proteomic profiles. PMID:26981549

  7. In situ imaging and proteome profiling indicate andrographolide is a highly promiscuous compound.

    PubMed

    Li, Lin; Wijaya, Hadhi; Samanta, Sanjay; Lam, Yulin; Yao, Shao Q

    2015-01-01

    Natural products represent an enormous source of pharmacologically useful compounds, and are often used as the starting point in modern drug discovery. Many biologically interesting natural products are however not being pursued as potential drug candidates, partly due to a lack of well-defined mechanism-of-action. Traditional in vitro methods for target identification of natural products based on affinity protein enrichment from crude cellular lysates cannot faithfully recapitulate protein-drug interactions in living cells. Reported herein are dual-purpose probes inspired by the natural product andrographolide, capable of both reaction-based, real-time bioimaging and in situ proteome profiling/target identification in live mammalian cells. Our results confirm that andrographolide is a highly promiscuous compound and engaged in covalent interactions with numerous previously unknown cellular targets in cell type-specific manner. We caution its potential therapeutic effects should be further investigated in detail. PMID:26105662

  8. Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems.

    PubMed

    Murray, Evan; Cho, Jae Hun; Goodwin, Daniel; Ku, Taeyun; Swaney, Justin; Kim, Sung-Yon; Choi, Heejin; Park, Young-Gyun; Park, Jeong-Yoon; Hubbert, Austin; McCue, Margaret; Vassallo, Sara; Bakh, Naveed; Frosch, Matthew P; Wedeen, Van J; Seung, H Sebastian; Chung, Kwanghun

    2015-12-01

    Combined measurement of diverse molecular and anatomical traits that span multiple levels remains a major challenge in biology. Here, we introduce a simple method that enables proteomic imaging for scalable, integrated, high-dimensional phenotyping of both animal tissues and human clinical samples. This method, termed SWITCH, uniformly secures tissue architecture, native biomolecules, and antigenicity across an entire system by synchronizing the tissue preservation reaction. The heat- and chemical-resistant nature of the resulting framework permits multiple rounds (>20) of relabeling. We have performed 22 rounds of labeling of a single tissue with precise co-registration of multiple datasets. Furthermore, SWITCH synchronizes labeling reactions to improve probe penetration depth and uniformity of staining. With SWITCH, we performed combinatorial protein expression profiling of the human cortex and also interrogated the geometric structure of the fiber pathways in mouse brains. Such integrated high-dimensional information may accelerate our understanding of biological systems at multiple levels. PMID:26638076

  9. Proteomic Profile of Brucella abortus-Infected Bovine Chorioallantoic Membrane Explants

    PubMed Central

    Mol, Juliana P. S.; Pires, Simone F.; Chapeaurouge, Alexander D.; Perales, Jonas; Santos, Renato L.; Andrade, Hélida M.; Lage, Andrey P.

    2016-01-01

    Brucella abortus is the etiological agent of bovine brucellosis, a zoonotic disease that causes significant economic losses worldwide. The differential proteomic profile of bovine chorioallantoic membrane (CAM) explants at early stages of infection with B. abortus (0.5, 2, 4, and 8 h) was determined. Analysis of CAM explants at 0.5 and 4 h showed the highest differences between uninfected and infected CAM explants, and therefore were used for the Differential Gel Electrophoresis (DIGE). A total of 103 spots were present in only one experimental group and were selected for identification by mass spectrometry (MALDI/ToF-ToF). Proteins only identified in extracts of CAM explants infected with B. abortus were related to recognition of PAMPs by TLR, production of reactive oxygen species, intracellular trafficking, and inflammation. PMID:27104343

  10. In situ imaging and proteome profiling indicate andrographolide is a highly promiscuous compound

    NASA Astrophysics Data System (ADS)

    Li, Lin; Wijaya, Hadhi; Samanta, Sanjay; Lam, Yulin; Yao, Shao Q.

    2015-06-01

    Natural products represent an enormous source of pharmacologically useful compounds, and are often used as the starting point in modern drug discovery. Many biologically interesting natural products are however not being pursued as potential drug candidates, partly due to a lack of well-defined mechanism-of-action. Traditional in vitro methods for target identification of natural products based on affinity protein enrichment from crude cellular lysates cannot faithfully recapitulate protein-drug interactions in living cells. Reported herein are dual-purpose probes inspired by the natural product andrographolide, capable of both reaction-based, real-time bioimaging and in situ proteome profiling/target identification in live mammalian cells. Our results confirm that andrographolide is a highly promiscuous compound and engaged in covalent interactions with numerous previously unknown cellular targets in cell type-specific manner. We caution its potential therapeutic effects should be further investigated in detail.

  11. Proteomic Profiling of Nonenzymatically Glycated Proteins in Human Plasma and Erythrocyte Membrane

    SciTech Connect

    Zhang, Qibin; Tang, Ning; Schepmoes, Athena A.; Phillips, Lawrence S.; Smith, Richard D.; Metz, Thomas O.

    2008-05-01

    Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. In this report, a thorough proteomic profiling of glycated proteins was attempted by using phenylboronate affinity chromatography to enrich glycated proteins and glycated, tryptic peptides from human plasma and erythrocyte membranes. Enriched peptides were subsequently analyzed by liquid chromatography coupled with electron transfer dissociation tandem mass spectrometry, and 76 and 31 proteins were confidently identified as glycated from human plasma and erythrocyte membrane, respectively. It was observed that most of the glycated proteins can be identified in samples from individuals with normal glucose tolerance, although samples from individuals with impaired glucose tolerance and type 2 diabetes mellitus have slightly higher numbers of glycated proteins and more glycation sites identified.

  12. MBPpred: Proteome-wide detection of membrane lipid-binding proteins using profile Hidden Markov Models.

    PubMed

    Nastou, Katerina C; Tsaousis, Georgios N; Papandreou, Nikos C; Hamodrakas, Stavros J

    2016-07-01

    A large number of modular domains that exhibit specific lipid binding properties are present in many membrane proteins involved in trafficking and signal transduction. These domains are present in either eukaryotic peripheral membrane or transmembrane proteins and are responsible for the non-covalent interactions of these proteins with membrane lipids. Here we report a profile Hidden Markov Model based method capable of detecting Membrane Binding Proteins (MBPs) from information encoded in their amino acid sequence, called MBPpred. The method identifies MBPs that contain one or more of the Membrane Binding Domains (MBDs) that have been described to date, and further classifies these proteins based on their position in respect to the membrane, either as peripheral or transmembrane. MBPpred is available online at http://bioinformatics.biol.uoa.gr/MBPpred. This method was applied in selected eukaryotic proteomes, in order to examine the characteristics they exhibit in various eukaryotic kingdoms and phyla. PMID:27048983

  13. In situ imaging and proteome profiling indicate andrographolide is a highly promiscuous compound

    PubMed Central

    Li, Lin; Wijaya, Hadhi; Samanta, Sanjay; Lam, Yulin; Yao, Shao Q.

    2015-01-01

    Natural products represent an enormous source of pharmacologically useful compounds, and are often used as the starting point in modern drug discovery. Many biologically interesting natural products are however not being pursued as potential drug candidates, partly due to a lack of well-defined mechanism-of-action. Traditional in vitro methods for target identification of natural products based on affinity protein enrichment from crude cellular lysates cannot faithfully recapitulate protein-drug interactions in living cells. Reported herein are dual-purpose probes inspired by the natural product andrographolide, capable of both reaction-based, real-time bioimaging and in situ proteome profiling/target identification in live mammalian cells. Our results confirm that andrographolide is a highly promiscuous compound and engaged in covalent interactions with numerous previously unknown cellular targets in cell type-specific manner. We caution its potential therapeutic effects should be further investigated in detail. PMID:26105662

  14. Proteomic profiling differences in serum from silicosis and chronic bronchitis patients: a comparative analysis

    PubMed Central

    Miao, Rongming; Ding, Bangmei; Zhang, Yingyi; Xia, Qian; Li, Yong

    2016-01-01

    Background Silicosis is a severe occupational disease characterized by pulmonary fibrosis, whereas chronic bronchitis (CB) is an acute inflammation of the airways. Differences in the mechanisms of pathogenesis of these diseases are not well understood, therefore we performed proteomic profiling of silicosis and CB patients and, compared the results. Methods Two-dimensional gel electrophoresis and MALDI-TOF-MS (matrix assisted laser desorption ionization time of flight mass spectrometry) were used to identify differentially accumulated proteins in stage I of silicosis (SI), stage II of silicosis (SII) and CB. Enzyme linked immunosorbent assay (ELISA) was employed to validate protein expression data. Results A total of 28 and 10 proteins were up- and down-regulated in SI, and 21 and 9 proteins were up- and down-regulated SII, compared with CB. Transforming growth factor beta-1 precursor and interferon beta precursor were up-regulated in CB, while interleukin 6, tumor necrosis factor (TNF) and a variant TNF receptor 13B were down-regulated in CB. Additionally, glycoprotein- and apolipoprotein-associated proteins including apolipoprotein A-IV and α-1-B-glycoprotein were up-regulated in CB, indicating an involvement in the pathogenesis of CB but not silicosis. By contrast, HLA-DRB1, medullasin and the proto-oncogene c-Fos were up-regulated in CB. Conclusions The immune, metabolism and apolipoprotein-related proteins were identified as playing specific and different roles in silicosis and CB. These proteomic profiling differences would facilitate further studies on the mechanisms underlying silicosis and CB, and may also prove useful to disease diagnosis and treatments. PMID:27076939

  15. Acclimation to different depths by the marine angiosperm Posidonia oceanica: transcriptomic and proteomic profiles

    PubMed Central

    Dattolo, Emanuela; Gu, Jenny; Bayer, Philipp E.; Mazzuca, Silvia; Serra, Ilia A.; Spadafora, Antonia; Bernardo, Letizia; Natali, Lucia; Cavallini, Andrea; Procaccini, Gabriele

    2013-01-01

    For seagrasses, seasonal and daily variations in light and temperature represent the mains factors driving their distribution along the bathymetric cline. Changes in these environmental factors, due to climatic and anthropogenic effects, can compromise their survival. In a framework of conservation and restoration, it becomes crucial to improve our knowledge about the physiological plasticity of seagrass species along environmental gradients. Here, we aimed to identify differences in transcriptomic and proteomic profiles, involved in the acclimation along the depth gradient in the seagrass Posidonia oceanica, and to improve the available molecular resources in this species, which is an important requisite for the application of eco-genomic approaches. To do that, from plant growing in shallow (−5 m) and deep (−25 m) portions of a single meadow, (i) we generated two reciprocal Expressed Sequences Tags (EST) libraries using a Suppressive Subtractive Hybridization (SSH) approach, to obtain depth/specific transcriptional profiles, and (ii) we identified proteins differentially expressed, using the highly innovative USIS mass spectrometry methodology, coupled with 1D-SDS electrophoresis and labeling free approach. Mass spectra were searched in the open source Global Proteome Machine (GPM) engine against plant databases and with the X!Tandem algorithm against a local database. Transcriptional analysis showed both quantitative and qualitative differences between depths. EST libraries had only the 3% of transcripts in common. A total of 315 peptides belonging to 64 proteins were identified by mass spectrometry. ATP synthase subunits were among the most abundant proteins in both conditions. Both approaches identified genes and proteins in pathways related to energy metabolism, transport and genetic information processing, that appear to be the most involved in depth acclimation in P. oceanica. Their putative rules in acclimation to depth were discussed. PMID:23785376

  16. Gender specificity of sucrose induced analgesia in human adults.

    PubMed

    Bhattacharjee, Manasi; Bhatia, Renu; Mathur, Rashmi

    2007-01-01

    Sweet, palatable substances such as sucrose are reported to calm infants undergoing routine investigative procedures. The analgesic effect persists in pre pubertal children and adults with a hint of gender dependent variation in the analgesic response. The present study was therefore designed to explore gender specificity of sucrose induced analgesia in adult volunteers utilizing the nociceptive flexion reflex, an objective tool for pain assessment. Nociceptive flexion reflex was recorded, both before and after (up to 15 min) ingestion of 100 ml of 25% sucrose solution in 6 male and 6 female volunteers. In the male volunteers the maximum amplitude of the response was 20.8 +/- 7.7 microV before sucrose ingestion and 22.6 +/- 9.1 microV, 6.6 +/- 0.7 microV, 6.2 +/- 1.1 microV, 7.5 +/- 0.9 microV at 0, 5, 10 and 15 minutes post sucrose ingestion respectively. In female volunteers, the maximum amplitude of the response was 33.7 +/- 17.7 microV before sucrose ingestion and 43.6 +/- 17.2 microV, 7.1 +/- 1.2 microV, 25.9 +/- 16.1 microV, 50.6 +/- 16.3 microV at the same time intervals post sucrose ingestion. The maximum amplitude values were significantly lower in the males at 10 and 15 minutes after sucrose ingestion (P < 0.05). This is the first objective report of gender specificity in sucrose induced analgesia in adult humans. The gender dependent variation in sucrose induced analgesia is prolonged in male (15 min) and short lived in female (5 min) volunteers. This knowledge may have important implications in pain management. PMID:18476396

  17. Gender-specific medicine in the genomic era.

    PubMed

    Legato, Marianne J

    2016-01-01

    This article is intended to illuminate several important changes in our concept of gender-specific medicine in the genomic era. It reviews the history of gender-specific medicine, pointing out the changes in our perception of the nature of biological sex and our expanding knowledge of how it affects the phenotype. The old debate about 'nature versus nurture' is now largely resolved; the two are inextricably intertwined as a result of epigenomic regulation of gene expression; many of the resulting phenotypic changes are inherited and affect future generations. More accurate, rapid and cheaper methods of editing genomic composition are implementing a more sophisticated understanding of how genes function and how individual components of the genome might be added or eliminated to maintain health and prevent disease. As Venter predicted, the new discipline of synthetic biology, based on the creation and use of novel 'designer' chromosomes is an inevitable expansion of our ability to decipher the naturally occurring genome and the factors that control its expression. As we move with unexpected and stunning rapidity into our exploration and manipulation of the genetic code, our investigations must acknowledge the solidly established fact that biological sex will have a profound impact on the interventions we have made and will make in the future. Unfortunately, in spite of the recent urging of the National Institutes of Health (NIH) that sex be included as an essential variable in all levels of scientific investigation, genuine issues remain to be resolved before all scientists accept not only the importance of doing this, but also how to implement it. PMID:26586840

  18. Comprehensive Analysis of the Triterpenoid Saponins Biosynthetic Pathway in Anemone flaccida by Transcriptome and Proteome Profiling

    PubMed Central

    Zhan, Chuansong; Li, Xiaohua; Zhao, Zeying; Yang, Tewu; Wang, Xuekui; Luo, Biaobiao; Zhang, Qiyun; Hu, Yanru; Hu, Xuebo

    2016-01-01

    Background: Anemone flaccida Fr. Shmidt (Ranunculaceae), commonly known as ‘Di Wu’ in China, is a perennial herb with limited distribution. The rhizome of A. flaccida has long been used to treat arthritis as a tradition in China. Studies disclosed that the plant contains a rich source of triterpenoid saponins. However, little is known about triterpenoid saponins biosynthesis in A. flaccida. Results: In this study, we conducted the tandem transcriptome and proteome profiling of a non-model medicinal plant, A. flaccida. Using Illumina HiSeq 2000 sequencing and iTRAQ technique, a total of 46,962 high-quality unigenes were obtained with an average sequence length of 1,310 bp, along with 1473 unique proteins from A. flaccida. Among the A. flaccida transcripts, 36,617 (77.97%) showed significant similarity (E-value < 1e-5) to the known proteins in the public database. Of the total 46,962 unigenes, 36,617 open reading frame (ORFs) were predicted. By the fragments per kilobases per million reads (FPKM) statistics, 14,004 isoforms/unigenes were found to be upregulated, and 14,090 isoforms/unigenes were down-regulated in the rhizomes as compared to those in the leaves. Based on the bioinformatics analysis, all possible enzymes involved in the triterpenoid saponins biosynthetic pathway of A. flaccida were identified, including cytosolic mevalonate pathway (MVA) and the plastidial methylerythritol pathway (MEP). Additionally, a total of 126 putative cytochrome P450 (CYP450) and 32 putative UDP glycosyltransferases were selected as the candidates of triterpenoid saponins modifiers. Among them, four of them were annotated as the gene of CYP716A subfamily, the key enzyme in the oleanane-type triterpenoid saponins biosynthetic pathway. Furthermore, based on RNA-Seq and proteome analysis, as well as quantitative RT-PCR verification, the expression level of gene and protein committed to triterpenoids biosynthesis in the leaf versus the rhizome was compared. Conclusion: A

  19. Comprehensive and quantitative proteomic analyses of zebrafish plasma reveals conserved protein profiles between genders and between zebrafish and human.

    PubMed

    Li, Caixia; Tan, Xing Fei; Lim, Teck Kwang; Lin, Qingsong; Gong, Zhiyuan

    2016-01-01

    Omic approaches have been increasingly used in the zebrafish model for holistic understanding of molecular events and mechanisms of tissue functions. However, plasma is rarely used for omic profiling because of the technical challenges in collecting sufficient blood. In this study, we employed two mass spectrometric (MS) approaches for a comprehensive characterization of zebrafish plasma proteome, i.e. conventional shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) for an overview study and quantitative SWATH (Sequential Window Acquisition of all THeoretical fragment-ion spectra) for comparison between genders. 959 proteins were identified in the shotgun profiling with estimated concentrations spanning almost five orders of magnitudes. Other than the presence of a few highly abundant female egg yolk precursor proteins (vitellogenins), the proteomic profiles of male and female plasmas were very similar in both number and abundance and there were basically no other highly gender-biased proteins. The types of plasma proteins based on IPA (Ingenuity Pathway Analysis) classification and tissue sources of production were also very similar. Furthermore, the zebrafish plasma proteome shares significant similarities with human plasma proteome, in particular in top abundant proteins including apolipoproteins and complements. Thus, the current study provided a valuable dataset for future evaluation of plasma proteins in zebrafish. PMID:27071722

  20. Comprehensive and quantitative proteomic analyses of zebrafish plasma reveals conserved protein profiles between genders and between zebrafish and human

    PubMed Central

    Li, Caixia; Tan, Xing Fei; Lim, Teck Kwang; Lin, Qingsong; Gong, Zhiyuan

    2016-01-01

    Omic approaches have been increasingly used in the zebrafish model for holistic understanding of molecular events and mechanisms of tissue functions. However, plasma is rarely used for omic profiling because of the technical challenges in collecting sufficient blood. In this study, we employed two mass spectrometric (MS) approaches for a comprehensive characterization of zebrafish plasma proteome, i.e. conventional shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) for an overview study and quantitative SWATH (Sequential Window Acquisition of all THeoretical fragment-ion spectra) for comparison between genders. 959 proteins were identified in the shotgun profiling with estimated concentrations spanning almost five orders of magnitudes. Other than the presence of a few highly abundant female egg yolk precursor proteins (vitellogenins), the proteomic profiles of male and female plasmas were very similar in both number and abundance and there were basically no other highly gender-biased proteins. The types of plasma proteins based on IPA (Ingenuity Pathway Analysis) classification and tissue sources of production were also very similar. Furthermore, the zebrafish plasma proteome shares significant similarities with human plasma proteome, in particular in top abundant proteins including apolipoproteins and complements. Thus, the current study provided a valuable dataset for future evaluation of plasma proteins in zebrafish. PMID:27071722

  1. Data for proteomic profiling of Anthers from a photosensitive male sterile mutant and wild-type cotton (Gossypium hirsutum L.).

    PubMed

    Liu, Ji; Pang, Chaoyou; Wei, Hengling; Song, Meizhen; Meng, Yanyan; Ma, Jianhui; Fan, Shuli; Yu, Shuxun

    2015-09-01

    Cotton is an important economic crop, used mainly for the production of textile fiber. Using a space mutation breeding technique, a novel photosensitive genetic male sterile mutant CCRI9106 was isolated from the wild-type upland cotton cultivar CCRI040029. To study the male sterile mechanisms of CCRI9106, histological and iTRAQ-facilitated proteomic analyses of anthers were performed. This data article contains data related to the research article titled iTRAQ-Facilitated Proteomic Profiling of Anthers From a Photosensitive Male Sterile Mutant and Wild-type Cotton (Gossypium hirsutum L.)[1]. This research article describes the iTRAQ-facilitated proteomic analysis of the wild-type and a photosensitive male sterile mutant in cotton. The report indicated that exine formation defect is the key reason for male sterility in mutant plant. The information presented here represents the tables and figures that detail the processing of the raw data obtained from iTRAQ analysis. PMID:26958592

  2. Profiling Lipid–protein Interactions Using Nonquenched Fluorescent Liposomal Nanovesicles and Proteome Microarrays*

    PubMed Central

    Lu, Kuan-Yi; Tao, Sheng-Ce; Yang, Tzu-Ching; Ho, Yu-Hsuan; Lee, Chia-Hsien; Lin, Chen-Ching; Juan, Hsueh-Fen; Huang, Hsuan-Cheng; Yang, Chin-Yu; Chen, Ming-Shuo; Lin, Yu-Yi; Lu, Jin-Ying; Zhu, Heng; Chen, Chien-Sheng

    2012-01-01

    Fluorescent liposomal nanovesicles (liposomes) are commonly used for lipid research and/or signal enhancement. However, the problem of self-quenching with conventional fluorescent liposomes limits their applications because these liposomes must be lysed to detect the fluorescent signals. Here, we developed a nonquenched fluorescent (NQF)1 liposome by optimizing the proportion of sulforhodamine B (SRB) encapsulant and lissamine rhodamine B-dipalmitoyl phosphatidylethanol (LRB-DPPE) on a liposomal surface for signal amplification. Our study showed that 0.3% of LRB-DPPE with 200 μm of SRB provided the maximal fluorescent signal without the need to lyse the liposomes. We also observed that the NQF liposomes largely eliminated self-quenching effects and produced greatly enhanced signals than SRB-only liposomes by 5.3-fold. To show their application in proteomics research, we constructed NQF liposomes that contained phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) and profiled its protein interactome using a yeast proteome microarray. Our profiling led to the identification of 162 PI(3,5)P2-specific binding proteins (PI(3,5)P2-BPs). We not only recovered many proteins that possessed known PI(3,5)P2-binding domains, but we also found two unknown Pfam domains (Pfam-B_8509 and Pfam-B_10446) that were enriched in our dataset. The validation of many newly discovered PI(3,5)P2-BPs was performed using a bead-based affinity assay. Further bioinformatics analyses revealed that the functional roles of 22 PI(3,5)P2-BPs were similar to those associated with PI(3,5)P2, including vesicle-mediated transport, GTPase, cytoskeleton, and kinase. Among the 162 PI(3,5)P2-BPs, we found a novel motif, HRDIKP[ES]NJLL that showed statistical significance. A docking simulation showed that PI(3,5)P2 interacted primarily with lysine or arginine side chains of the newly identified PI(3,5)P2-binding kinases. Our study showed that this new tool would greatly benefit profiling lipid

  3. Proteome and Transcriptome Profiles of a Her2/Neu-driven Mouse Model of Breast Cancer

    SciTech Connect

    Schoenherr, Regine M.; Kelly-Spratt, Karen S.; Lin, Chen Wei; Whiteaker, Jeffrey R.; Liu, Tao; Holzman, Ted; Coleman, Ilsa; Feng, Li-Chia; Lorentzen, Travis D.; Krasnoselsky, Alexei L.; Wang, Pei; Liu, Yan; Gurley, Kay E.; Amon, Lynn M.; Schepmoes, Athena A.; Moore, Ronald J.; Camp, David G.; Chodosh, Lewis A.; Smith, Richard D.; Nelson, Peter S.; McIntosh, Martin; Kemp, Christopher; Paulovich, Amanda G.

    2011-04-01

    In recent years, mouse models have proven to be invaluable in expanding our understanding of cancer biology. We have amassed a tremendous amount of proteomics and transcriptomics data profiling blood and tissues from a Her2-driven mouse model of breast cancer that closely recapitulates the pathology and natural history of human breast cancer. The purpose of this report is to make all of these data publicly available in raw and processed forms, as a resource to the community. Importantly, high quality biospecimens from this same mouse model are freely available through a sample repository that we established, so researchers can readily obtain samples to test biological hypotheses without the need of breeding animals and collecting biospecimens. Specifically, six proteomics and six transcriptomics datasets are available, with the former encompassing 841 liquid chromatography-tandem mass spectrometry (LC-MS/MS) experiments of both plasma and tissue samples, and the latter including 255 individual microarray analyses of five different tissue types (thymus, spleen, liver, blood cells, and breast ± laser capture microdissection). A total of 18,880 unique peptides were identified with a PeptideProphet error rate ≤1%, with 3884 non-redundant protein groups identified in five plasma datasets, and 1659 non-redundant protein groups in a tissue dataset (4977 non-redundant protein groups in total). We anticipate that these data will be of use to the community for software tool development, investigations of analytical variation in MS/MS data, development of quality control tools (multiple technical replicates are provided for a subset of the data), empirical selection of proteotypic peptides for multiple reaction monitoring mass spectrometry, and for advancing our understanding of cancer biology.

  4. Quantitative Proteomic Profiling of Early and Late Responses to Salicylic Acid in Cucumber Leaves

    PubMed Central

    Li, Liang; Shang, Qing-Mao

    2016-01-01

    Salicylic acid (SA) is an important phytohormone that plays vital regulatory roles in plant growth, development, and stress responses. However, studies on the molecular mechanism of SA, especially during the early SA responses, are lagging behind. In this study, we initiated a comprehensive isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic analysis to explore the early and late SA-responsive proteins in leaves of cucumber (Cucumis sativus L.) seedlings. Upon SA application through the roots, endogenous SA accumulated in cucumber leaves. By assaying the changes in marker gene expression and photosynthetic rate, we collected samples at 12 h and 72 h post treatment (hpt) to profile the early and late SA responsiveness, respectively. The iTRAQ assay followed by tandem mass spectrometry revealed 135 differentially expressed proteins (DEPs) at 12 hpt and 301 DEPs at 72 hpt. The functional categories for these SA-responsive proteins included in a variety of biochemical processes, including photosynthesis, redox homeostasis, carbohydrate and energy metabolism, lipid metabolism, transport, protein folding and modification, proteolysis, cell wall organization, and the secondary phenylpropanoid pathway. Conclusively, based on the abundant changes of these DEPs, together with their putative functions, we proposed a possible SA-responsive protein network. It appears that SA could elicit reactive oxygen species (ROS) production via enhancing the photosynthetic electron transferring, and then confer some growth-promoting and stress-priming effects on cells during the late phase, including enhanced photosynthesis and ROS scavenging, altered carbon metabolic flux for the biosynthesis of amino acids and nucleotides, and cell wall reorganization. Overall, the present iTRAQ assay provides higher proteome coverage and deepened our understanding of the molecular basis of SA-responses. PMID:27551830

  5. Establishment of a proteome profile and identification of molecular markers for mouse spermatogonial stem cells

    PubMed Central

    Zhou, Quan; Guo, Yueshuai; Zheng, Bo; Shao, Binbin; Jiang, Min; Wang, Gaigai; Zhou, Tao; Wang, Lei; Zhou, Zuomin; Guo, Xuejiang; Huang, Xiaoyan

    2015-01-01

    Spermatogonial stem cells (SSCs) are undifferentiated cells that are required to maintain spermatogenesis throughout the reproductive life of mammals. Although SSC transplantation and culture provide a powerful tool to identify the mechanisms regulating SSC function, the precise signalling mechanisms governing SSC self-renewal and specific surface markers for purifying SSCs remain to be clearly determined. In the present study, we established a steady SSC culture according to the method described by Shinohara's lab. Fertile progeny was produced after transplantation of cultured SSCs into infertile mouse testis, and the red fluorescence exhibited by the culture cell membranes was stably and continuously transmitted to the offspring. Next, via advanced mass spectrometry and an optimized proteomics platform, we constructed the proteome profile, with 682 proteins expressed in SSCs. Furthermore bioinformatics analysis showed that the list contained several known molecules that are regulated in SSCs. Several nucleoproteins and membrane proteins were chosen for further exploration using immunofluorescence and RT-PCR. The results showed that SALL1, EZH2, and RCOR2 are possibly involved in the self-renewal mechanism of SSCs. Furthermore, the results of tissue-specific expression analysis showed that Gpat2 and Pld6 were uniquely and highly expressed in mouse testes and cultured SSCs. The cellular localization of PLD6 was further explored and the results showed it was primarily expressed in the spermatogonial membrane of mouse testes and cultured SSCs. The proteins identified in this study form the basis for further exploring the molecular mechanism of self-renewal in SSCs and for identifying specific surface markers of SSCs. PMID:25352495

  6. Quantitative Proteomic Profiling of Early and Late Responses to Salicylic Acid in Cucumber Leaves.

    PubMed

    Dong, Chun-Juan; Cao, Ning; Li, Liang; Shang, Qing-Mao

    2016-01-01

    Salicylic acid (SA) is an important phytohormone that plays vital regulatory roles in plant growth, development, and stress responses. However, studies on the molecular mechanism of SA, especially during the early SA responses, are lagging behind. In this study, we initiated a comprehensive isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic analysis to explore the early and late SA-responsive proteins in leaves of cucumber (Cucumis sativus L.) seedlings. Upon SA application through the roots, endogenous SA accumulated in cucumber leaves. By assaying the changes in marker gene expression and photosynthetic rate, we collected samples at 12 h and 72 h post treatment (hpt) to profile the early and late SA responsiveness, respectively. The iTRAQ assay followed by tandem mass spectrometry revealed 135 differentially expressed proteins (DEPs) at 12 hpt and 301 DEPs at 72 hpt. The functional categories for these SA-responsive proteins included in a variety of biochemical processes, including photosynthesis, redox homeostasis, carbohydrate and energy metabolism, lipid metabolism, transport, protein folding and modification, proteolysis, cell wall organization, and the secondary phenylpropanoid pathway. Conclusively, based on the abundant changes of these DEPs, together with their putative functions, we proposed a possible SA-responsive protein network. It appears that SA could elicit reactive oxygen species (ROS) production via enhancing the photosynthetic electron transferring, and then confer some growth-promoting and stress-priming effects on cells during the late phase, including enhanced photosynthesis and ROS scavenging, altered carbon metabolic flux for the biosynthesis of amino acids and nucleotides, and cell wall reorganization. Overall, the present iTRAQ assay provides higher proteome coverage and deepened our understanding of the molecular basis of SA-responses. PMID:27551830

  7. Proteomic profile of KSR1-regulated signalling in response to genotoxic agents in breast cancer.

    PubMed

    Zhang, Hua; Angelopoulos, Nicos; Xu, Yichen; Grothey, Arnhild; Nunes, Joao; Stebbing, Justin; Giamas, Georgios

    2015-06-01

    Kinase suppressor of Ras 1 (KSR1) has been implicated in tumorigenesis in multiple cancers, including skin, pancreatic and lung carcinomas. However, our recent study revealed a role of KSR1 as a tumour suppressor in breast cancer, the expression of which is potentially correlated with chemotherapy response. Here, we aimed to further elucidate the KSR1-regulated signalling in response to genotoxic agents in breast cancer. Stable isotope labelling by amino acids in cell culture (SILAC) coupled to high-resolution mass spectrometry (MS) was implemented to globally characterise cellular protein levels induced by KSR1 in the presence of doxorubicin or etoposide. The acquired proteomic signature was compared and GO-STRING analysis was subsequently performed to illustrate the activated functional signalling networks. Furthermore, the clinical associations of KSR1 with identified targets and their relevance in chemotherapy response were examined in breast cancer patients. We reveal a comprehensive repertoire of thousands of proteins identified in each dataset and compare the unique proteomic profiles as well as functional connections modulated by KSR1 after doxorubicin (Doxo-KSR1) or etoposide (Etop-KSR1) stimulus. From the up-regulated top hits, several proteins, including STAT1, ISG15 and TAP1 are also found to be positively associated with KSR1 expression in patient samples. Moreover, high KSR1 expression, as well as high abundance of these proteins, is correlated with better survival in breast cancer patients who underwent chemotherapy. In aggregate, our data exemplify a broad functional network conferred by KSR1 with genotoxic agents and highlight its implication in predicting chemotherapy response in breast cancer. PMID:26022350

  8. Proteomic profile of carbonylated proteins in rat liver: discovering possible mechanisms for tetracycline-induced steatosis.

    PubMed

    Deng, Zhenglu; Yan, Siyu; Hu, Hui; Duan, Zhigui; Yin, Lanxuan; Liao, Shenke; Sun, Yubai; Yin, Dazhong; Li, Guolin

    2015-01-01

    To investigate biochemical mechanisms for the tetracycline-induced steatosis in rats, targeted proteins of oxidative modification were profiled. The results showed that tetracycline induced lipid accumulation, oxidative stress, and cell viability decline in HepG2 cells only under the circumstances of palmitic acid overload. Tetracycline administration in rats led to significant decrement in blood lipids, while resulted in more than four times increment in intrahepatic triacylglycerol and typical microvesicular steatosis in the livers. The triacylglycerol levels were positively correlated with oxidative stress. Proteomic profiles of carbonylated proteins revealed 26 targeted proteins susceptible to oxidative modification and most of them located in mitochondria. Among them, the long-chain specific acyl-CoA dehydrogenase was one of the key enzymes regulating fatty acid β-oxidation. Oxidative modification of the enzyme in the tetracycline group depressed its enzymatic activity. In conclusion, the increased influx of lipid into the livers is the first hit of tetracycline-induced microvesicular steatosis. Oxidative stress is an essential part of the second hit, which may arise from the lipid overload and attack a series of functional proteins, aggravating the development of steatosis. The 26 targeted proteins revealed here provide a potential direct link between oxidative stress and tetracycline-induced steatosis. PMID:25332112

  9. Differential lysine acetylation profiles of Erwinia amylovora strains revealed by proteomics

    PubMed Central

    Wu, Xia; Vellaichamy, Adaikkalam; Wang, Dongping; Zamdborg, Leonid; Kelleher, Neil L.; Huber, Steven C.; Zhao, Youfu

    2015-01-01

    Protein lysine acetylation (LysAc) has recently been demonstrated to be widespread in E. coli and Salmonella, and to broadly regulate bacterial physiology and metabolism. However, LysAc in plant pathogenic bacteria is largely unknown. Here we first report the lysine acetylome of Erwinia amylovora, an enterobacterium causing serious fire blight disease of apples and pears. Immunoblots using generic anti-lysine acetylation antibodies demonstrated that growth conditions strongly affected the LysAc profiles in E. amylovora. Differential LysAc profiles were also observed for two E. amylovora strains, known to have differential virulence in plants, indicating translational modification of proteins may be important in determining virulence of bacterial strains. Proteomic analysis of LysAc in two E. amylovora strains identified 141 LysAc sites in 96 proteins that function in a wide range of biological pathways. Consistent with previous reports, 44% of the proteins are involved in metabolic processes, including central metabolism, lipopolysaccharide, nucleotide and amino acid metabolism. Interestingly, for the first time, several proteins involved in E. amylovora virulence, including exopolysaccharide amylovoran biosynthesis- and type III secretion-associated proteins, were found to be lysine acetylated, suggesting that LysAc may play a major role in bacterial virulence. Comparative analysis of LysAc sites in E. amylovora and E. coli further revealed the sequence and structural commonality for LysAc in the two organisms. Collectively, these results reinforce the notion that LysAc of proteins is widespread in bacterial metabolism and virulence. PMID:23234799

  10. Differential lysine acetylation profiles of Erwinia amylovora strains revealed by proteomics.

    PubMed

    Wu, Xia; Vellaichamy, Adaikkalam; Wang, Dongping; Zamdborg, Leonid; Kelleher, Neil L; Huber, Steven C; Zhao, Youfu

    2013-02-21

    Protein lysine acetylation (LysAc) has recently been demonstrated to be widespread in E. coli and Salmonella, and to broadly regulate bacterial physiology and metabolism. However, LysAc in plant pathogenic bacteria is largely unknown. Here we first report the lysine acetylome of Erwinia amylovora, an enterobacterium causing serious fire blight disease of apples and pears. Immunoblots using generic anti-lysine acetylation antibodies demonstrated that growth conditions strongly affected the LysAc profiles in E. amylovora. Differential LysAc profiles were also observed for two E. amylovora strains, known to have differential virulence in plants, indicating translational modification of proteins may be important in determining virulence of bacterial strains. Proteomic analysis of LysAc in two E. amylovora strains identified 141 LysAc sites in 96 proteins that function in a wide range of biological pathways. Consistent with previous reports, 44% of the proteins are involved in metabolic processes, including central metabolism, lipopolysaccharide, nucleotide and amino acid metabolism. Interestingly, for the first time, several proteins involved in E. amylovora virulence, including exopolysaccharide amylovoran biosynthesis- and type III secretion-associated proteins, were found to be lysine acetylated, suggesting that LysAc may play a major role in bacterial virulence. Comparative analysis of LysAc sites in E. amylovora and E. coli further revealed the sequence and structural commonality for LysAc in the two organisms. Collectively, these results reinforce the notion that LysAc of proteins is widespread in bacterial metabolism and virulence. PMID:23234799

  11. Proteomics profiling of cholangiocarcinoma exosomes: A potential role of oncogenic protein transferring in cancer progression.

    PubMed

    Dutta, Suman; Reamtong, Onrapak; Panvongsa, Wittaya; Kitdumrongthum, Sarunya; Janpipatkul, Keatdamrong; Sangvanich, Polkit; Piyachaturawat, Pawinee; Chairoungdua, Arthit

    2015-09-01

    Cholangiocarcinoma (CCA), a common primary malignant tumor of bile duct epithelia, is highly prevalent in Asian countries and unresponsive to chemotherapeutic drugs. Thus, a newly recognized biological entity for early diagnosis and treatment is highly needed. Exosomes are small membrane bound vesicles found in body fluids and released by most cell types including cancer cells. The vesicles contain specific subset of proteins and nucleic acids corresponding to cell types and play essential roles in pathophysiological processes. The present study aimed to assess the protein profiles of CCA-derived exosomes and their potential roles. We have isolated exosomes from CCA cells namely KKU-M213 and KKU-100 derived from Thai patients and their roles were investigated by incubation with normal human cholangiocyte (H69) cells. Exosomes were internalized into H69 cells and had no effects on viability or proliferation of the host cells. Interestingly, the exosomes from KKU-M213 cells only induced migration and invasion of H69 cells. Proteomic analysis of the exosomes from KKU-M213 cells disclosed multiple cancer related proteins that are not present in H69 exosomes. Consistent with the protein profile, treatment with KKU-M213 exosomes induced β-catenin and reduced E-cadherin expressions in H69 cells. Collectively, our results suggest that a direct cell-to-cell transfer of oncogenic proteins via exosomal pathway may be a novel mechanism for CCA progression and metastasis. PMID:26148937

  12. Comparative analysis of proteomic profiles between endometrial caruncular and intercaruncular areas in ewes during the peri-implantation period

    PubMed Central

    2013-01-01

    The endometrium of sheep consists of plenty of raised aglandular areas called caruncular (C), and intensely glandular intercaruncular areas (IC). In order to better understand the endometrium involved mechanisms of implantation, we used LC-MS/MS technique to profile the proteome of ovine endometrial C areas and IC areas separately during the peri-implantation period, and then compared the proteomic profiles between these two areas. We successfully detected 1740 and 1813 proteins in C areas and IC areas respectively. By comparing the proteome of these two areas, we found 170 differentially expressed proteins (DEPs) (P < 0.05), functional bioinformatics analysis showed these DEPs were mainly involved in growth and remodeling of endometrial tissue, cell adhesion and protein transport, and so on. Our study, for the first time, provided a proteomic reference for elucidating the differences between C and IC areas, as an integrated function unit respectively, during the peri-implantation period. The results could help us to better understand the implantation in the ewes. In addition, we established a relatively detailed protein database of ovine endometrium, which provide a unique reference for further studies. PMID:24093944

  13. Proteomic Profiling of Paraffin-Embedded Samples Identifies Metaplasia-Specific and Early-Stage Gastric Cancer Biomarkers

    PubMed Central

    Sousa, Josane F.; Ham, Amy-Joan L.; Whitwell, Corbin; Nam, Ki Taek; Lee, Hyuk-Joon; Yang, Han-Kwang; Kim, Woo Ho; Zhang, Bing; Li, Ming; LaFleur, Bonnie; Liebler, Daniel C.; Goldenring, James R.

    2013-01-01

    Early diagnosis and curative resection are the predominant factors associated with increased survival in patients with gastric cancer. However, most gastric cancer cases are still diagnosed at later stages. Since most pathologic specimens are archived as FFPE samples, the ability to use them to generate expression profiles can greatly improve cancer biomarker discovery. We sought to uncover new biomarkers for stomach preneoplastic metaplasias and neoplastic lesions by generating proteome profiles using FFPE samples. We combined peptide isoelectric focusing and liquid chromatography–tandem mass spectrometry analysis to generate proteomic profiles from FFPE samples of intestinal-type gastric cancer, metaplasia, and normal mucosa. The expression patterns of selected proteins were analyzed by immunostaining first in single tissue sections from normal stomach, metaplasia, and gastric cancer and later in larger tissue array cohorts. We detected 60 proteins up-regulated and 87 proteins down-regulated during the progression from normal mucosa to metaplasia to gastric cancer. Two of the up-regulated proteins, LTF and DMBT1, were validated as specific markers for spasmolytic polypeptide–expressing metaplasia and intestinal metaplasia, respectively. In cancers, significantly lower levels of DMBT1 or LTF correlated with more advanced disease and worse prognosis. Thus, proteomic profiling using FFPE samples has led to the identification of two novel markers for stomach metaplasias and gastric cancer prognosis. PMID:22944598

  14. No clinical benefit of gender-specific total knee arthroplasty

    PubMed Central

    Zhu, Chen; Wang, Jiaxing; Cheng, Mengqi; Peng, Xiaochun; Wang, Qi; Zhang, Xianlong

    2014-01-01

    Background and purpose There is no consensus regarding the clinical relevance of gender-specific prostheses in total knee arthroplasty (TKA). We summarize the current best evidence in a comparison of clinical and radiographic outcomes between gender-specific prostheses and standard unisex prostheses in female patients. Methods We used the PubMed, Embase, Cochrane, Science Citation Index, and Scopus databases. We included randomized controlled trials published up to January 2013 that compared gender-specific prostheses with standard unisex prostheses in female patients who underwent primary TKAs. Results 6 trials involving 423 patients with 846 knee joints met the inclusion criteria. No statistically significant differences were observed between the 2 designs regarding pain, range of motion (ROM), knee scores, satisfaction, preference, complications, and radiographic results. The gender-specific design (Gender Solutions; Zimmer Inc, Warsaw, Indiana) reduced the prevalence of overhang. However, it had less overall coverage of the femoral condyles compared to the unisex group. In fact, the femoral prosthesis in the standard unisex group matched better than that in the gender-specific group. Interpretation Gender-specific prostheses do not appear to confer any benefit in terms of clinician- and patient-reported outcomes for the female knee. PMID:24954488

  15. Proteome-derived Peptide Libraries to Study the Substrate Specificity Profiles of Carboxypeptidases*

    PubMed Central

    Tanco, Sebastian; Lorenzo, Julia; Garcia-Pardo, Javier; Degroeve, Sven; Martens, Lennart; Aviles, Francesc Xavier; Gevaert, Kris; Van Damme, Petra

    2013-01-01

    Through processing peptide and protein C termini, carboxypeptidases participate in the regulation of various biological processes. Few tools are however available to study the substrate specificity profiles of these enzymes. We developed a proteome-derived peptide library approach to study the substrate preferences of carboxypeptidases. Our COFRADIC-based approach takes advantage of the distinct chromatographic behavior of intact peptides and the proteolytic products generated by the action of carboxypeptidases, to enrich the latter and facilitate its MS-based identification. Two different peptide libraries, generated either by chymotrypsin or by metalloendopeptidase Lys-N, were used to determine the substrate preferences of human metallocarboxypeptidases A1 (hCPA1), A2 (hCPA2), and A4 (hCPA4). In addition, our approach allowed us to delineate the substrate specificity profile of mouse mast cell carboxypeptidase (MC-CPA or mCPA3), a carboxypeptidase suggested to function in innate immune responses regulation and mast cell granule homeostasis, but which thus far lacked a detailed analysis of its substrate preferences. mCPA3 was here shown to preferentially remove bulky aromatic amino acids, similar to hCPA2. This was also shown by a hierarchical cluster analysis, grouping hCPA1 close to hCPA4 in terms of its P1 primed substrate specificity, whereas hCPA2 and mCPA3 cluster separately. The specificity profile of mCPA3 may further aid to elucidate the function of this mast cell carboxypeptidase and its biological substrate repertoire. Finally, we used this approach to evaluate the substrate preferences of prolylcarboxypeptidase, a serine carboxypeptidase shown to cleave C-terminal amino acids linked to proline and alanine. PMID:23620545

  16. Lens proteome map and alpha-crystallin profile of the catfish Rita rita.

    PubMed

    Mohanty, Bimal Prasanna; Bhattacharjee, Soma; Das, Manas Kumar

    2011-02-01

    Crystallins are a diverse group of proteins that constitute nearly 90% of the total soluble proteins of the vertebrate eye lens and these tightly packed crystallins are responsible for transparency of the lens. These proteins have been studied in different model and non-model species for understanding the modifications they undergo with ageing that lead to cataract, a disease of protein aggregation. In the present investigation, we studied the lens crystallin profile of the tropical freshwater catfish Rita rita. Profiles of lens crystallins were analyzed and crystallin proteome maps of Rita rita were generated for the first time. alphaA-crystallins, member of the alpha-crystallin family, which are molecular chaperons and play crucial role in maintaining lens transparency were identified by 1- and 2-D immunoblot analysis with anti-alphaA-crystallin antibody. Two protein bands of 19-20 kDa were identified as alphaA-crystallins on 1-D immunoblots and these bands separated into 10 discrete spots on 2-D immunoblot. However, anti-alphaB-crystallin and antiphospho-alphaB-crystallin antibodies were not able to detect any immunoreactive bands on 1- and 2-D immunoblots, indicating alphaB-crystallin was either absent or present in extremely low concentration in Rita rita lens. Thus, Rita rita alpha-crystallins are more like that of the catfish Clarias batrachus and the mammal kangaroo in its alphaA- and alphaB-crystallin content (contain low amount from 5-9% of alphaB-crystallin) and unlike the dogfish, zebrafish, human, bovine and mouse alpha-crystallins (contain higher amount of alphaB-crystallin from 25% in mouse and bovine to 85% in dogfish). Results of the present study can be the baseline information for stimulating further investigation on Rita rita lens crystallins for comparative lens proteomics. Comparing and contrasting the alpha-crystallins of the dogfish and Rita rita may provide valuable information on the functional attributes of alphaA- and alphaB-isoforms, as

  17. Protein alterations associated with pancreatic cancer and chronic pancreatitis found in human plasma using global quantitative proteomics profiling

    PubMed Central

    Pan, Sheng; Chen, Ru; Crispin, David A.; May, Damon; Stevens, Tyler; McIntosh, Martin; Bronner, Mary P.; Ziogas, Argyrios; Anton-Culver, Hoda; Brentnall, Teresa A.

    2011-01-01

    Pancreatic cancer is a lethal disease that is difficult to diagnose at early stages when curable treatments are effective. Biomarkers that can improve current pancreatic cancer detection would have great value in improving patient management and survival rate. A large scale quantitative proteomics study was performed to search for the plasma protein alterations associated with pancreatic cancer. The enormous complexity of the plasma proteome and the vast dynamic range of protein concentration therein present major challenges for quantitative global profiling of plasma. To address these challenges, multi-dimensional fractionation at both protein and peptide levels was applied to enhance the depth of proteomics analysis. Employing stringent criteria, more than thirteen hundred proteins total were identified in plasma across 8-orders of magnitude in protein concentration. Differential proteins associated with pancreatic cancer were identified, and their relationship with the proteome of pancreatic tissue and pancreatic juice from our previous studies was discussed. A subgroup of differentially expressed proteins was selected for biomarker testing using an independent cohort of plasma and serum samples from well-diagnosed patients with pancreatic cancer, chronic pancreatitis and non-pancreatic disease controls. Using ELISA methodology, the performance of each of these protein candidates was benchmarked against CA19-9, the current gold standard for a pancreatic cancer blood test. A composite marker of TIMP1 and ICAM1 demonstrate significantly better performance than CA19-9 in distinguishing pancreatic cancer from the non-pancreatic disease controls and chronic pancreatitis controls. In addition, protein AZGP1 was identified as a biomarker candidate for chronic pancreatitis. The discovery and technical challenges associated with plasma-based quantitative proteomics are discussed and may benefit the development of plasma proteomics technology in general. The protein

  18. Quantitative Proteome Profiling of Street Rabies Virus-Infected Mouse Hippocampal Synaptosomes.

    PubMed

    Sun, Xiaoning; Shi, Ning; Li, Ying; Dong, Chunyan; Zhang, Maolin; Guan, Zhenhong; Duan, Ming

    2016-09-01

    It is well established now that neuronal dysfunction rather than structural damage may be responsible for the development of rabies. In order to explore the underlying mechanisms in rabies virus (RABV) and synaptic dysfunctions, a quantitative proteome profiling was carried out on synaptosome samples from mice hippocampus. Synaptosome samples from mice hippocampus were isolated and confirmed by Western blot and transmission electron microscopy. Synaptosome protein content changes were quantitatively detected by Nano-LC-MS/MS. Protein functions were classified by the Gene Ontology (GO) and KEGG pathway. PSICQUIC was used to create a network. MCODE algorithm was applied to obtain subnetworks. Of these protein changes, 45 were upregulated and 14 were downregulated following RABV infection relative to non-infected (mock) synaptosomes. 28 proteins were unique to mock treatment and 12 were unique to RABV treatment. Proteins related to metabolism and synaptic vesicle showed the most changes in expression levels. Furthermore, protein-protein interaction (PPI) networks revealed that several key biological processes related to synaptic functions potentially were modulated by RABV, including energy metabolism, cytoskeleton organization, and synaptic transmission. These data will be useful for better understanding of neuronal dysfunction of rabies and provide the foundation for future research. PMID:27155843

  19. Proteomic profiling of small-molecule inhibitors reveals dispensability of MTH1 for cancer cell survival.

    PubMed

    Kawamura, Tatsuro; Kawatani, Makoto; Muroi, Makoto; Kondoh, Yasumitsu; Futamura, Yushi; Aono, Harumi; Tanaka, Miho; Honda, Kaori; Osada, Hiroyuki

    2016-01-01

    Since recent publications suggested that the survival of cancer cells depends on MTH1 to avoid incorporation of oxidized nucleotides into the cellular DNA, MTH1 has attracted attention as a potential cancer therapeutic target. In this study, we identified new purine-based MTH1 inhibitors by chemical array screening. However, although the MTH1 inhibitors identified in this study targeted cellular MTH1, they exhibited only weak cytotoxicity against cancer cells compared to recently reported first-in-class inhibitors. We performed proteomic profiling to investigate the modes of action by which chemically distinct MTH1 inhibitors induce cancer cell death, and found mechanistic differences among the first-in-class MTH1 inhibitors. In particular, we identified tubulin as the primary target of TH287 and TH588 responsible for the antitumor effects despite the nanomolar MTH1-inhibitory activity in vitro. Furthermore, overexpression of MTH1 did not rescue cells from MTH1 inhibitor-induced cell death, and siRNA-mediated knockdown of MTH1 did not suppress cancer cell growth. Taken together, we conclude that the cytotoxicity of MTH1 inhibitors is attributable to off-target effects and that MTH1 is not essential for cancer cell survival. PMID:27210421

  20. Proteomic profiling of small-molecule inhibitors reveals dispensability of MTH1 for cancer cell survival

    PubMed Central

    Kawamura, Tatsuro; Kawatani, Makoto; Muroi, Makoto; Kondoh, Yasumitsu; Futamura, Yushi; Aono, Harumi; Tanaka, Miho; Honda, Kaori; Osada, Hiroyuki

    2016-01-01

    Since recent publications suggested that the survival of cancer cells depends on MTH1 to avoid incorporation of oxidized nucleotides into the cellular DNA, MTH1 has attracted attention as a potential cancer therapeutic target. In this study, we identified new purine-based MTH1 inhibitors by chemical array screening. However, although the MTH1 inhibitors identified in this study targeted cellular MTH1, they exhibited only weak cytotoxicity against cancer cells compared to recently reported first-in-class inhibitors. We performed proteomic profiling to investigate the modes of action by which chemically distinct MTH1 inhibitors induce cancer cell death, and found mechanistic differences among the first-in-class MTH1 inhibitors. In particular, we identified tubulin as the primary target of TH287 and TH588 responsible for the antitumor effects despite the nanomolar MTH1-inhibitory activity in vitro. Furthermore, overexpression of MTH1 did not rescue cells from MTH1 inhibitor–induced cell death, and siRNA-mediated knockdown of MTH1 did not suppress cancer cell growth. Taken together, we conclude that the cytotoxicity of MTH1 inhibitors is attributable to off-target effects and that MTH1 is not essential for cancer cell survival. PMID:27210421

  1. Proteomic profiling of cardiac tissue by isolation of nuclei tagged in specific cell types (INTACT)

    PubMed Central

    Amin, Nirav M.; Greco, Todd M.; Kuchenbrod, Lauren M.; Rigney, Maggie M.; Chung, Mei-I; Wallingford, John B.; Cristea, Ileana M.; Conlon, Frank L.

    2014-01-01

    The proper dissection of the molecular mechanisms governing the specification and differentiation of specific cell types requires isolation of pure cell populations from heterogeneous tissues and whole organisms. Here, we describe a method for purification of nuclei from defined cell or tissue types in vertebrate embryos using INTACT (isolation of nuclei tagged in specific cell types). This method, previously developed in plants, flies and worms, utilizes in vivo tagging of the nuclear envelope with biotin and the subsequent affinity purification of the labeled nuclei. In this study we successfully purified nuclei of cardiac and skeletal muscle from Xenopus using this strategy. We went on to demonstrate the utility of this approach by coupling the INTACT approach with liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic methodologies to profile proteins expressed in the nuclei of developing hearts. From these studies we have identified the Xenopus orthologs of 12 human proteins encoded by genes, which when mutated in human lead to congenital heart disease. Thus, by combining these technologies we are able to identify tissue-specific proteins that are expressed and required for normal vertebrate organ development. PMID:24496632

  2. Deciphering the proteomic profile of rice (Oryza sativa) bran: a pilot study.

    PubMed

    Ferrari, Fabio; Fumagalli, Marco; Profumo, Antonella; Viglio, Simona; Sala, Alberto; Dolcini, Lorenzo; Temporini, Caterina; Nicolis, Stefania; Merli, Daniele; Corana, Federica; Casado, Begona; Iadarola, Paolo

    2009-12-01

    The exact knowledge of the qualitative and quantitative protein components of rice bran is an essential aspect to be considered for a better understanding of the functional properties of this resource. Aim of the present investigation was to extract the largest number of rice bran proteins and to obtain their qualitative characterization. For this purpose, three different extraction protocols have been applied either on full-fat or on defatted rice bran. Likewise, to identify the highest number of proteins, MS data collected from 1-DE, 2-DE and gel-free procedures have been combined. These approaches allowed to unambiguously identify 43 proteins that were classified as signalling/regulation proteins (30%), proteins with enzymatic activity (30%), storage proteins (30%), transfer (5%) and structural (5%) proteins. The fact that all extraction and identification procedures have been performed in triplicate with an excellent reproducibility provides a rationale for considering the platform of proteins shown in this study as the potential proteome profile of rice bran. It also represents a source of information to evaluate better the qualities of rice bran as food resource. PMID:19960476

  3. Proteomic profiling of human sera for discovery of potential biomarkers to monitor abstinence from alcohol abuse

    PubMed Central

    Lai, Xianyin; Liangpunsakul, Suthat; Li, Kaigang; Witzmann, Frank A.

    2015-01-01

    Although numerous biomarkers or biomarker candidates have been discovered to detect levels of drinking and intervals of time after last drinking episode, only a few biomarkers have been applied to monitor abstinence in a longer interval (≥ 6 weeks) from alcohol abuse. Considering sample sources, sensitivity, and specificity, new biomarkers from blood with better accuracy are needed. To address this, serum proteomic profiles were compared between pre- and post- treatment samples from subjects seeking treatment for alcohol abuse and dependence in an intensive 6-week daily outpatient program using high-abundance plasma protein immunodepletion and LC-MS/MS techniques. Protein identification, quantification, candidate biomarker selection, and prioritization analyses were carried out. Among the 246 quantified serum proteins, abundance of 13 and 45 proteins in female and male subjects were significantly changed (p ≤ 0.05), respectively. Of these biomarker candidate proteins, 2 (female) and 8 (male) proteins were listed in category 1, with high area under the receiver operating characteristic (ROC) curve (AUC), sensitivity, specificity, and fold change. In summary, several new biomarker candidates have been identified to monitor abstinence from alcohol abuse. PMID:25475211

  4. Proteomic profiling of halloysite clay nanotube exposure in intestinal cell co-culture

    PubMed Central

    Lai, Xianyin; Agarwal, Mangilal; Lvov, Yuri M.; Pachpande, Chetan; Varahramyan, Kody; Witzmann, Frank A.

    2013-01-01

    Halloysite is aluminosilicate clay with a hollow tubular structure with nanoscale internal and external diameters. Assessment of halloysite biocompatibility has gained importance in view of its potential application in oral drug delivery. To investigate the effect of halloysite nanotubes on an in vitro model of the large intestine, Caco-2/HT29-MTX cells in monolayer co-culture were exposed to nanotubes for toxicity tests and proteomic analysis. Results indicate that halloysite exhibits a high degree of biocompatibility characterized by an absence of cytotoxicity, in spite of elevated pro-inflammatory cytokine release. Exposure-specific changes in expression were observed among 4081 proteins analyzed. Bioinformatic analysis of differentially expressed protein profiles suggest that halloysite stimulates processes related to cell growth and proliferation, subtle responses to cell infection, irritation and injury, enhanced antioxidant capability, and an overall adaptive response to exposure. These potentially relevant functional effects warrant further investigation in in vivo models and suggest that chronic or bolus occupational exposure to halloysite nanotubes may have unintended outcomes. PMID:23606564

  5. Proteomics profiling reveals novel proteins and functions of the plant stigma exudate

    PubMed Central

    Rejón, Juan David; Delalande, François; Castro, Antonio Jesús

    2013-01-01

    Proteomic analysis of the stigmatic exudate of Lilium longiflorum and Olea europaea led to the identification of 51 and 57 proteins, respectively, most of which are described for the first time in this secreted fluid. These results indicate that the stigmatic exudate is an extracellular environment metabolically active, participating in at least 80 different biological processes and 97 molecular functions. The stigma exudate showed a markedly catabolic profile and appeared to possess the enzyme machinery necessary to degrade large polysaccharides and lipids secreted by papillae to smaller units, allowing their incorporation into the pollen tube during pollination. It may also regulate pollen-tube growth in the pistil through the selective degradation of tube-wall components. Furthermore, some secreted proteins were involved in pollen-tube adhesion and orientation, as well as in programmed cell death of the papillae cells in response to either compatible pollination or incompatible pollen rejection. Finally, the results also revealed a putative cross-talk between genetic programmes regulating stress/defence and pollination responses in the stigma. PMID:24151302

  6. Proteomic profiling of halloysite clay nanotube exposure in intestinal cell co-culture.

    PubMed

    Lai, Xianyin; Agarwal, Mangilal; Lvov, Yuri M; Pachpande, Chetan; Varahramyan, Kody; Witzmann, Frank A

    2013-11-01

    Halloysite is aluminosilicate clay with a hollow tubular structure with nanoscale internal and external diameters. Assessment of halloysite biocompatibility has gained importance in view of its potential application in oral drug delivery. To investigate the effect of halloysite nanotubes on an in vitro model of the large intestine, Caco-2/HT29-MTX cells in monolayer co-culture were exposed to nanotubes for toxicity tests and proteomic analysis. Results indicate that halloysite exhibits a high degree of biocompatibility characterized by an absence of cytotoxicity, in spite of elevated pro-inflammatory cytokine release. Exposure-specific changes in expression were observed among 4081 proteins analyzed. Bioinformatic analysis of differentially expressed protein profiles suggest that halloysite stimulates processes related to cell growth and proliferation, subtle responses to cell infection, irritation and injury, enhanced antioxidant capability, and an overall adaptive response to exposure. These potentially relevant functional effects warrant further investigation in in vivo models and suggest that chronic or bolus occupational exposure to halloysite nanotubes may have unintended outcomes. PMID:23606564

  7. The effect of colostrum intake on blood plasma proteome profile in newborn lambs: low abundance proteins

    PubMed Central

    2014-01-01

    Background Colostrum intake by newborn lambs plays a fundamental role in the perinatal period, ensuring lamb survival. In this study, blood plasma samples from two groups of newborn lambs (Colostrum group and Delayed Colostrum group) at 2 and 14 h after birth were treated to reduce the content of high abundance proteins and analyzed using Two-Dimensional Differential in Gel Electrophoresis and MALDI MS/MS for protein identification in order to investigate low abundance proteins with immune function in newborn lambs. Results The results showed that four proteins were increased in the blood plasma of lambs due to colostrum intake. These proteins have not been previously described as increased in blood plasma of newborn ruminants by colostrum intake. Moreover, these proteins have been described as having an immune function in other species, some of which were previously identified in colostrum and milk. Conclusions In conclusion, colostrum intake modified the low abundance proteome profile of blood plasma from newborn lambs, increasing the concentration of apolipoprotein A-IV, plasminogen, serum amyloid A and fibrinogen, demonstrating that colostrum is essential, not only for the provision of immunoglobulins, but also because of increases in several low abundance proteins with immune function. PMID:24708841

  8. Proteomic profiling of 13 paired ductal infiltrating breast carcinomas and non-tumoral adjacent counterparts.

    PubMed

    Pucci-Minafra, Ida; Cancemi, Patrizia; Marabeti, Maria Rita; Albanese, Nadia Ninfa; Di Cara, Gianluca; Taormina, Pietra; Marrazzo, Antonio

    2007-01-01

    According to recent statistics, breast cancer remains one of the leading causes of death among women in Western countries. Breast cancer is a complex and heterogeneous disease, presently classified into several subtypes according to their cellular origin. Among breast cancer histotypes, infiltrating ductal carcinoma represents the most common and potentially aggressive form. Despite the current progress achieved in early cancer detection and treatment, including the new generation of molecular therapies, there is still need for identification of multiparametric biomarkers capable of discriminating between cancer subtypes and predicting cancer progression for personalized therapies. One established step in this direction is the proteomic strategy, expected to provide enough information on breast cancer profiling. To this aim, in the present study we analyzed 13 breast cancer tissues and their matched non-tumoral tissues by 2-DE. Collectively, we identified 51 protein spots, corresponding to 34 differentially expressed proteins, which may represent promising candidate biomarkers for molecular-based diagnosis of breast cancer and for pattern discovery. The relevance of these proteins as factors contributing to breast carcinogenesis is discussed. PMID:21136615

  9. Genomic and proteomic profiling of oxidative stress response in human diploid fibroblasts

    PubMed Central

    Xie, Lifang; Pandey, Ritu; Xu, Beibei; Tsaprailis, George; Chen, Qin M.

    2016-01-01

    A number of lines of evidence suggest that senescence of normal human diploid fibroblasts (HDFs) in culture is relevant to the process of aging in vivo. Using normal human skin diploid fibroblasts, we examine the changes in genes and proteins following treatment with a mild dose of H2O2, which induces premature senescence. Multidimensional Protein Identification Technology (MudPIT) in combination with mass spectrometry analyses of whole cell lysates from HDFs detected 65 proteins in control group, 48 proteins in H2O2-treated cells and 109 proteins common in both groups. In contrast, cDNA microarray analyses show 173 genes up-regulated and 179 genes down-regulated upon H2O2 treatment. Both MudPIT and cDNA microarray analyses indicate that H2O2 treatment caused elevated levels of thioredoxin reductase 1. Semi-quantitative RT-PCR and Western-blot were able to verify the finding. Out of a large number of genes or proteins detected, only a small fraction shows the overlap between the outcomes of microarray versus proteomics. The low overlap suggests the importance of considering proteins instead of transcripts when investigating the gene expression profile altered by oxidative stress. PMID:18654835

  10. Towards the profiling of the Arabidopsis thaliana plasma membrane transportome by targeted proteomics.

    PubMed

    Monneuse, Jean-Marc; Sugano, Madeleine; Becue, Thierry; Santoni, Véronique; Hem, Sonia; Rossignol, Michel

    2011-05-01

    Plant membranes bear a variety of transporters belonging to multigene families that are affected by environmental and nutritional conditions. In addition, they often display high-sequence identity, making difficult in-depth investigation by current shot-gun strategies. In this study, we set up a targeted proteomics approach aimed at identifying and quantifying within single experiments the five major proton pumps of the autoinhibited H(+) ATPases (AHA) family, the 13 plasma membrane intrinsic proteins (PIP) water channels (PIPs), and ten members of ammonium transporters (AMTs) and nitrate transporter (NRT) families. Proteotypic peptides were selected and isotopically labeled heavy versions were used for technical optimization and for quantification of the corresponding light version in biological samples. This approach allowed to quantify simultaneously nine PIPs in leaf membranes and 13 PIPs together with three autoinhibited H(+) ATPases, two ammonium transporters, and two NRTs in root membranes. Similarly, it was used to investigate the effect of a salt stress on the expression of these latter 20 transporters in roots. These novel isoform-specific data were compared with published transcriptome information and revealed a close correlation between PIP isoforms and transcripts levels. The obtained resource is reusable and can be expanded to other transporter families for large-scale profiling of membrane transporters. PMID:21413151

  11. Proteomic and Glycoproteomic Profilings Reveal That Post-translational Modifications of Toxins Contribute to Venom Phenotype in Snakes.

    PubMed

    Andrade-Silva, Débora; Zelanis, André; Kitano, Eduardo S; Junqueira-de-Azevedo, Inácio L M; Reis, Marcelo S; Lopes, Aline S; Serrano, Solange M T

    2016-08-01

    Snake venoms are biological weapon systems composed of secreted proteins and peptides that are used for immobilizing or killing prey. Although post-translational modifications are widely investigated because of their importance in many biological phenomena, we currently still have little understanding of how protein glycosylation impacts the variation and stability of venom proteomes. To address these issues, here we characterized the venom proteomes of seven Bothrops snakes using a shotgun proteomics strategy. Moreover, we compared the electrophoretic profiles of native and deglycosylated venoms and, in order to assess their subproteomes of glycoproteins, we identified the proteins with affinity for three lectins with different saccharide specificities and their putative glycosylation sites. As proteinases are abundant glycosylated toxins, we examined the effect of N-deglycosylation on their catalytic activities and show that the proteinases of the seven venoms were similarly affected by removal of N-glycans. Moreover, we prospected putative glycosylation sites of transcripts of a B. jararaca venom gland data set and detected toxin family related patterns of glycosylation. Based on our global analysis, we report that Bothrops venom proteomes and glycoproteomes contain a core of components that markedly define their composition, which is conserved upon evolution in parallel to other molecular markers that determine their phylogenetic classification. PMID:27297130

  12. High abundance synovial fluid proteome: distinct profiles in health and osteoarthritis

    PubMed Central

    Gobezie, Reuben; Kho, Alvin; Krastins, Bryan; Sarracino, David A; Thornhill, Thomas S; Chase, Michael; Millett, Peter J; Lee, David M

    2007-01-01

    The development of increasingly high-throughput and sensitive mass spectroscopy-based proteomic techniques provides new opportunities to examine the physiology and pathophysiology of many biologic fluids and tissues. The purpose of this study was to determine protein expression profiles of high-abundance synovial fluid (SF) proteins in health and in the prevalent joint disease osteoarthritis (OA). A cross-sectional study of 62 patients with early OA (n = 21), patients with late OA (n = 21), and control individuals (n = 20) was conducted. SF proteins were separated by using one-dimensional PAGE, and the in-gel digested proteins were analyzed by electrospray ionization tandem mass spectrometry. A total of 362 spots were examined and 135 high-abundance SF proteins were identified as being expressed across all three study cohorts. A total of 135 SF proteins were identified. Eighteen proteins were found to be significantly differentially expressed between control individuals and OA patients. Two subsets of OA that are not dependent on disease duration were identified using unsupervised analysis of the data. Several novel SF proteins were also identified. Our analyses demonstrate no disease duration-dependent differences in abundant protein composition of SF in OA, and we clearly identified two previously unappreciated yet distinct subsets of protein profiles in this disease cohort. Additionally, our findings reveal novel abundant protein species in healthy SF whose functional contribution to SF physiology was not previously recognized. Finally, our studies identify candidate biomarkers for OA with potential for use as highly sensitive and specific tests for diagnostic purposes or for evaluating therapeutic response. PMID:17407561

  13. Proteomic profiling in multiple sclerosis clinical courses reveals potential biomarkers of neurodegeneration.

    PubMed

    Liguori, Maria; Qualtieri, Antonio; Tortorella, Carla; Direnzo, Vita; Bagalà, Angelo; Mastrapasqua, Mariangela; Spadafora, Patrizia; Trojano, Maria

    2014-01-01

    The aim of our project was to perform an exploratory analysis of the cerebrospinal fluid (CSF) proteomic profiles of Multiple Sclerosis (MS) patients, collected in different phases of their clinical course, in order to investigate the existence of peculiar profiles characterizing the different MS phenotypes. The study was carried out on 24 Clinically Isolated Syndrome (CIS), 16 Relapsing Remitting (RR) MS, 11 Progressive (Pr) MS patients. The CSF samples were analysed using the Matrix Assisted Laser Desorption Ionisation Time Of Flight (MALDI-TOF) mass spectrometer in linear mode geometry and in delayed extraction mode (m/z range: 1000-25000 Da). Peak lists were imported for normalization and statistical analysis. CSF data were correlated with demographic, clinical and MRI parameters. The evaluation of MALDI-TOF spectra revealed 348 peak signals with relative intensity ≥ 1% in the study range. The peak intensity of the signals corresponding to Secretogranin II and Protein 7B2 were significantly upregulated in RRMS patients compared to PrMS (p<0.05), whereas the signals of Fibrinogen and Fibrinopeptide A were significantly downregulated in CIS compared to PrMS patients (p<0.04). Additionally, the intensity of the Tymosin β4 peak was the only signal to be significantly discriminated between the CIS and RRMS patients (p = 0.013). Although with caution due to the relatively small size of the study populations, and considering that not all the findings remained significant after adjustment for multiple comparisons, in our opinion this mass spectrometry evaluation confirms that this technique may provide useful and important information to improve our understanding of the complex pathogenesis of MS. PMID:25098164

  14. Proteomic Profiling of Exosomes Leads to the Identification of Novel Biomarkers for Prostate Cancer

    SciTech Connect

    Duijvesz, Diederick; Burnum-Johnson, Kristin E.; Gritsenko, Marina A.; Hoogland, Marije; Vredenbregt-van den Berg, Mirella S.; Willemsen, Rob; Luider, Theo N.; Pasa-Tolic, Ljiljana; Jenster, Guido

    2013-12-31

    Introduction: Current markers for prostate cancer, such as PSA lack specificity. Therefore, novel biomarkers are needed. Unfortunately, biomarker discovery from body fluids is often hampered by the high abundance of many proteins unrelated to disease. An attractive alternative biomarker discovery approach is the isolation of small vesicles (exosomes, ~100 nm). They contain proteins that are specific to the tissue from which they are derived and therefore can be considered as treasure chests for disease-specific marker discovery. Profiling prostate cancer-derived exosomes could reveal new markers for this malignancy. Materials and Methods: Exosomes were isolated from 2 immortalized primary prostate epithelial cells (PNT2C2 and RWPE-1) and 2 PCa cell lines (PC346C and VCaP) by ultracentrifugation. Proteomic analyses utilized a nanoLC coupled with an LTQ-Orbitrap operated in tandem MS (MS/MS) mode, followed by the Accurate Mass and Time (AMT) tag approach. Exosomal proteins were validated by Western blotting. A Tissue Micro Array, containing 481 different PCa samples (radical prostatectomy), was used to correlate candidate markers with several clinical-pathological parameters such as PSA, Gleason score, biochemical recurrence, and (PCa-related) death. Results: Proteomic characterization resulted in the identification of 263 proteins by at least 2 peptides. Specifically analysis of exosomes from PNT2C2, RWPE-1, PC346C, and VCaP identified 248, 233, 169, and 216 proteins, respectively. Statistical analyses revealed 52 proteins differently expressed between PCa and control cells, 9 of which were more abundant in PCa. Validation by Western blotting confirmed a higher abundance of FASN, XPO1 and PDCD6IP (ALIX) in PCa exosomes. The Tissue Micro 4 Array showed strong correlation of higher Gleason scores and local recurrence with increased cytoplasmic XPO1 (P<0.001). Conclusions: Differentially abundant proteins of cell line-derived exosomes make a clear subdivision between

  15. Protein profiling of mefloquine resistant Plasmodium falciparum using mass spectrometry-based proteomics

    PubMed Central

    Reamtong, Onrapak; Srimuang, Krongkan; Saralamba, Naowarat; Sangvanich, Polkit; Day, Nicholas P.J.; White, Nicholas J.; Imwong, Mallika

    2015-01-01

    Malaria is a mosquito borne infectious disease caused by protozoa of genus Plasmodium. There are five species of Plasmodium that are found to infect humans. Plasmodium falciparum can cause severe malaria leading to higher morbidity and mortality of malaria than the other four species. Antimalarial resistance is the major obstacle to control malaria. Mefloquine was used in combination with Artesunate for uncomplicated P. falciparum in South East Asia and it has developed and established mefloquine resistance in this region. Here, gel-enhanced liquid chromatography/tandem mass spectrometry (GeLC–MS/MS)-based proteomics and label-free quantification were used to explore the protein profiles of mefloquine-sensitive and -induced resistant P. falciparum. A Thai P. falciparum isolate (S066) was used as a model in this research. Our data revealed for the first time that 69 proteins exhibited at least 2-fold differences in their expression levels between the two parasite lines. Of these, 36 were up-regulated and 33 were down-regulated in the mefloquine-resistant line compared with the mefloquine-sensitive line. These findings are consistent with those of past studies, where the multidrug resistance protein Pgh1 showed an up-regulation pattern consistent with that expected from its average 3-copy pfmdr1 gene number. Pgh1 and eight other up-regulated proteins (i.e., histo-aspartyl protease protein, exportin 1, eukaryotic translation initiation factor 3 subunit 8, peptidyl-prolyl cis-trans isomerase, serine rich protein homologue, exported protein 1, ATP synthase beta chain and phospholipid scramblase 1) were further validated for their expression levels using reverse transcriptase quantitative real-time PCR. The data support the up-regulation status in the mefloquine-resistant parasite line of all the candidate genes referred to above. Therefore, GeLC–MS/MS-based proteomics combined with label-free quantification is a reliable approach for exploring mefloquine resistance

  16. A brain proteome profile in rats exposed to methylmercury or thimerosal (ethylmercury).

    PubMed

    de Oliveira Souza, Vanessa Cristina; de Marco, Kátia Cristina; Laure, Hélen Julie; Rosa, José Cesar; Barbosa, Fernando

    2016-01-01

    Exposure to organomercurials has been associated with harmful effects on the central nervous system (CNS). However, the mechanisms underlying organomercurial-mediated neurotoxic effects need to be elucidated. Exposure to toxic elements may promote cellular modifications such as alterations in protein synthesis in an attempt to protect tissues and organs from damage. In this context, the use of a "proteomic profile" is an important tool to identify potential early biomarkers or targets indicative of neurotoxicity. The aim of this study was to investigate potential modifications in rat cerebral cell proteome following exposure to methylmercury (MeHg) or ethylmercury (EtHg). For MeHg exposure, animals were administered by gavage daily 140 µg/kg/d of Hg (as MeHg) for 60 d and sacrificed 24 h after the last treatment. For EtHg exposure, 800 µg/kg/d of Hg (as EtHg) was given intramuscularly (im) in a single dose and rats were sacrificed after 4 h. Control groups received saline either by gavage or im. After extraction of proteins from whole brain samples and separation by two-dimensional electrophoresis (2-DE), 26 differentially expressed proteins were identified from exposed animals by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF/TOF). Both MeHg and EtHg exposure induced an overexpression of calbindin, a protein that acts as a neuroprotective agent by (1) adjusting the concentration of Ca(2+) within cells and preventing neurodegenerative diseases and (2) decreasing expression of glutamine synthetase, a crucial protein involved in regulation of glutamate concentration in synaptic cleft. In contrast, expression of superoxide dismutase (SOD), a protein involved in antioxidant defense, was elevated in brain of MeHg-exposed animals. Taken together, our data provide new valuable information on the possible molecular mechanisms associated with MeHg- and EtHg-mediated toxicity in cerebral tissue. These observed protein alterations may be considered as

  17. Protein profiling of human lung telocytes and microvascular endothelial cells using iTRAQ quantitative proteomics

    PubMed Central

    Zheng, Yonghua; Cretoiu, Dragos; Yan, Guoquan; Cretoiu, Sanda Maria; Popescu, Laurentiu M; Fang, Hao; Wang, Xiangdong

    2014-01-01

    Telocytes (TCs) are described as a particular type of cells of the interstitial space (www.telocytes.com). Their main characteristics are the very long telopodes with alternating podoms and podomers. Recently, we performed a comparative proteomic analysis of human lung TCs with fibroblasts, demonstrating that TCs are clearly a distinct cell type. Therefore, the present study aims to reinforce this idea by comparing lung TCs with endothelial cells (ECs), since TCs and ECs share immunopositivity for CD34. We applied isobaric tag for relative and absolute quantification (iTRAQ) combined with automated 2-D nano-ESI LC-MS/MS to analyse proteins extracted from TCs and ECs in primary cell cultures. In total, 1609 proteins were identified in cell cultures. 98 proteins (the 5th day), and 82 proteins (10th day) were confidently quantified (screened by two-sample t-test, P < 0.05) as up- or down-regulated (fold change >2). We found that in TCs there are 38 up-regulated proteins at the 5th day and 26 up-regulated proteins at the 10th day. Bioinformatics analysis using Panther revealed that the 38 proteins associated with TCs represented cellular functions such as intercellular communication (via vesicle mediated transport) and structure morphogenesis, being mainly cytoskeletal proteins and oxidoreductases. In addition, we found 60 up-regulated proteins in ECs e.g.: cell surface glycoprotein MUC18 (15.54-fold) and von Willebrand factor (5.74-fold). The 26 up-regulated proteins in TCs at 10th day, were also analysed and confirmed the same major cellular functions, while the 56 down-regulated proteins confirmed again their specificity for ECs. In conclusion, we report here the first extensive comparison of proteins from TCs and ECs using a quantitative proteomics approach. Our data show that TCs are completely different from ECs. Protein expression profile showed that TCs play specific roles in intercellular communication and intercellular signalling. Moreover, they might

  18. In-Depth Analysis of a Plasma or Serum Proteome Using a 4D Protein Profiling Method

    PubMed Central

    Tang, Hsin-Yao; Beer, Lynn A.; Speicher, David W.

    2011-01-01

    Comprehensive proteomic analysis of human plasma or serum has been a major strategy used to identify biomarkers that serve as indicators of disease. However, such in-depth proteomic analyses are challenging due to the complexity and extremely large dynamic range of protein concentrations in plasma. Therefore, reduction in sample complexity through multidimensional pre-fractionation strategies is critical, particularly for the detection of low-abundance proteins that have the potential to be the most specific disease biomarkers. We describe here a 4D protein profiling method that we developed for comprehensive proteomic analyses of both plasma and serum. Our method consists of abundant protein depletion coupled with separation strategies – microscale solution isoelectrofocusing and 1D SDS-PAGE – followed by reversed-phase separation of tryptic peptides prior to LC–MS/MS. Using this profiling strategy, we routinely identify a large number of proteins over nine orders of magnitude, including a substantial number of proteins at the low ng/mL or lower levels from approximately 300 μL of plasma sample. PMID:21468940

  19. Proteomic protease specificity profiling of clostridial collagenases reveals their intrinsic nature as dedicated degraders of collagen☆☆☆

    PubMed Central

    Eckhard, Ulrich; Huesgen, Pitter F.; Brandstetter, Hans; Overall, Christopher M.

    2014-01-01

    Clostridial collagenases are among the most efficient degraders of collagen. Most clostridia are saprophytes and secrete proteases to utilize proteins in their environment as carbon sources; during anaerobic infections, collagenases play a crucial role in host colonization. Several medical and biotechnological applications have emerged utilizing their high collagenolytic efficiency. However, the contribution of the functionally most important peptidase domain to substrate specificity remains unresolved. We investigated the active site sequence specificity of the peptidase domains of collagenase G and H from Clostridium histolyticum and collagenase T from Clostridium tetani. Both prime and non-prime cleavage site specificity were simultaneously profiled using Proteomic Identification of protease Cleavage Sites (PICS), a mass spectrometry-based method utilizing database searchable proteome-derived peptide libraries. For each enzyme we identified > 100 unique-cleaved peptides, resulting in robust cleavage logos revealing collagen-like specificity patterns: a strong preference for glycine in P3 and P1′, proline at P2 and P2′, and a slightly looser specificity at P1, which in collagen is typically occupied by hydroxyproline. This specificity for the classic collagen motifs Gly-Pro-X and Gly-X-Hyp represents a remarkable adaptation considering the complex requirements for substrate unfolding and presentation that need to be fulfilled before a single collagen strand becomes accessible for cleavage. Biological significance We demonstrate the striking sequence specificity of a family of clostridial collagenases using proteome derived peptide libraries and PICS, Proteomic Identification of protease Cleavage Sites. In combination with the previously published crystal structures of these proteases, our results represent an important piece of the puzzle in understanding the complex mechanism underlying collagen hydrolysis, and pave the way for the rational design of

  20. Differential proteomic profiling reveals regulatory proteins and novel links between primary metabolism and spinosad production in Saccharopolyspora spinosa

    PubMed Central

    2014-01-01

    Background Saccharopolyspora spinosa is an important producer of antibiotic spinosad with clarified biosynthesis pathway but its complex regulation networks associated with primary metabolism and secondary metabolites production almost have never been concerned or studied before. The proteomic analysis of a novel Saccharopolyspora spinosa CCTCC M206084 was performed and aimed to provide a global profile of regulatory proteins. Results Two-dimensional-liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 1090, 1166, 701, and 509 proteins from four phases respectively, i.e., the logarithmic growth phase (T1), early stationary phase (T2), late stationary phase (T3), and decline phase (T4). Among the identified proteins, 1579 were unique to the S. spinosa proteome, including almost all the enzymes for spinosad biosynthesis. Trends in protein expression over the various time phases were deduced from using the modified protein abundance index (PAI), revealed the importance of stress pathway proteins and other global regulatory network proteins during spinosad biosynthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis followed by one-dimensional LC-MS/MS identification revealed similar trend of protein expression from four phases with the results of semi-quantification by PAI. qRT-PCR analysis revealed that 6 different expressed genes showed a positive correlation between changes at translational and transcriptional expression level. Expression of three proteins that likely promote spinosad biosynthesis, namely, 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase (MHSM), glutamine synthetase (GS) and cyclic nucleotide-binding domain-containing protein (CNDP) was validated by western blot, which confirmed the results of proteomic analysis. Conclusions This study is the first systematic analysis of the S. spinosa proteome during fermentation and its valuable proteomic data of regulatory proteins may be used to enhance

  1. Mitochondrial proteome: toward the detection and profiling of disease associated alterations.

    PubMed

    Herrmann, Paul C; Herrmann, E Clifford

    2012-01-01

    Existing at the heart of cellular energy metabolism, the mitochondrion is uniquely positioned to have a major impact on human disease processes. Examples of mitochondrial impact on human pathology abound and include etiologies ranging from inborn errors of metabolism to the site of activity of a variety of toxic compounds. In this review, the unique aspects of the mechanisms related to the mitochondrial proteome are discussed along with an overview of the literature related to mitochondrial proteomic exploration. The review includes discussion of potential areas for exploration and advantages of applying proteomic techniques to the study of mitochondria. PMID:22081351

  2. Effects of stem cell therapy on protein profile of parkinsonian rats using an(18) O-labeling quantitative proteomic approach.

    PubMed

    Liu, Yahui; Liu, Kefu; Qin, Wei; Liu, Chenghao; Zheng, Xiaowei; Deng, Yulin; Qing, Hong

    2016-03-01

    The application of neural stem cell (NSC) research to neurodegenerative diseases has led to promising clinical trials. Currently, NSC therapy is most promising for Parkinson's disease (PD). We conducted behavioral tests and immunoassays for the profiling of a PD model in rats to assess the therapeutic effects of NSC treatments. Further, using a multiple sample comparison workflow, combined with (18) O-labeled proteome mixtures, we compared the differentially expressed proteins from control, PD, and NSC-treated PD rats. The results were analyzed bioinformatically and verified by Western blot. Based on our initial findings, we believe that the proteomic approach is a valuable tool in evaluating the therapeutic effects of NSC transplantation on neurodegenerative disorders. PMID:26791447

  3. Proteomic profiling of Rhipicephalus (Boophilus) microplus midgut responses to infection with Babesia bovis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Differences in protein expression in midgut tissue of uninfected and Babesia bovis-infected southern cattle ticks, Rhipicephalus (Boophilus) microplus, were investigated in an effort to establish a proteome database containing proteins involved in successful pathogen transmission. The electrophoreti...

  4. Mass Spectrometry-based Proteomics: Qualitative Identification to Activity-based Protein Profiling

    PubMed Central

    Cardoza, Job D.; Parikh, Jignesh R.; Ficarro, Scott B.; Marto, Jarrod A.

    2011-01-01

    Mass spectrometry has become the method of choice for proteome characterization, including multi-component protein complexes (typically tens to hundreds of proteins) and total protein expression (up to tens of thousands of proteins), in biological samples. Qualitative sequence assignment based on MS/MS spectra is relatively well-defined, while statistical metrics for relative quantification have not completely stabilized. Nonetheless, proteomics studies have progressed to the point whereby various gene-, pathway-, or network-oriented computational frameworks may be used to place mass spectrometry data into biological context. Despite this progress, the dynamic range of protein expression remains a significant hurdle, and impedes comprehensive proteome analysis. Methods designed to enrich specific protein classes have emerged as an effective means to characterize enzymes or other catalytically active proteins that are otherwise difficult to detect in typical discovery mode proteomics experiments. Collectively, these approaches will facilitate identification of biomarkers and pathways relevant to diagnosis and treatment of human disease. PMID:22231900

  5. Dysbindin as a novel biomarker for pancreatic ductal adenocarcinoma identified by proteomic profiling.

    PubMed

    Guo, Xin; Lv, Xiaohui; Fang, Cheng; Lv, Xing; Wang, Fengsong; Wang, Dongmei; Zhao, Jun; Ma, Yueyun; Xue, Yu; Bai, Quan; Yao, Xuebiao; Chen, Yong

    2016-10-15

    Pancreatic adenocarcinoma (PDAC) is known to have a poor prognosis partly because of lack of effective biomarkers. In the test set, we investigated dysbindin (DTNBP1) as a potential biomarker for PDAC by comparing preoperative and postoperative serum mass spectrometry (MS) proteomic profilings. Of the included 50 PDAC patients, 42 (positivity of 84.0%) detected a lower MS peak in postoperative serums than preoperative ones which was then identified as dysbindin. In the verification set, receiver operating characteristics (ROC) were used to assess diagnostic efficiency. 550 participants were included in the verification set [250 with PDAC, 80 with benign biliary obstruction (BBO), 70 with chronic pancreatitis (CP) and 150 healthy donors (HD)]. Dysbindin was increased in PDAC patient sera than in all controls. ROC curves revealed the optimum diagnostic cutoff for dysbindin was 699.16 pg/ml [area under curve (AUC) 0.849 (95% CI 0.812-0.885), sensitivity 81.9% and specificity 84.7%]. Raised concentration of dysbindin in sera could differentiate PDAC from BBO, CP and HD. Moreover, dysbindin maintained its diagnostic accuracy for PDAC patients who were CA19-9 negative [AUC 0.875 (95% CI 0.804-0.945), sensitivity 83.0%, specificity 89.0%] and for patients with benign biliary obstruction [AUC 0.849 (95% CI 0.803-0.894), sensitivity 82.3%, specificity 84.0%].Our discovery of dysbindin may complement measurement of CA19-9 in the diagnosis of PDAC and help to discriminate PDAC from other pancreatic diseases or begin biliary obstruction. PMID:27281120

  6. Proteomic Profiling and Identification of Immunodominant Spore Antigens of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis‡

    PubMed Central

    DelVecchio, Vito G.; Connolly, Joseph P.; Alefantis, Timothy G.; Walz, Alexander; Quan, Marian A.; Patra, Guy; Ashton, John M.; Whittington, Jessica T.; Chafin, Ryan D.; Liang, Xudong; Grewal, Paul; Khan, Akbar S.; Mujer, Cesar V.

    2006-01-01

    Differentially expressed and immunogenic spore proteins of the Bacillus cereus group of bacteria, which includes Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis, were identified. Comparative proteomic profiling of their spore proteins distinguished the three species from each other as well as the virulent from the avirulent strains. A total of 458 proteins encoded by 232 open reading frames were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis for all the species. A number of highly expressed proteins, including elongation factor Tu (EF-Tu), elongation factor G, 60-kDa chaperonin, enolase, pyruvate dehydrogenase complex, and others exist as charge variants on two-dimensional gels. These charge variants have similar masses but different isoelectric points. The majority of identified proteins have cellular roles associated with energy production, carbohydrate transport and metabolism, amino acid transport and metabolism, posttranslational modifications, and translation. Novel vaccine candidate proteins were identified using B. anthracis polyclonal antisera from humans postinfected with cutaneous anthrax. Fifteen immunoreactive proteins were identified in B. anthracis spores, whereas 7, 14, and 7 immunoreactive proteins were identified for B. cereus and in the virulent and avirulent strains of B. thuringiensis spores, respectively. Some of the immunodominant antigens include charge variants of EF-Tu, glyceraldehyde-3-phosphate dehydrogenase, dihydrolipoamide acetyltransferase, Δ-1-pyrroline-5-carboxylate dehydrogenase, and a dihydrolipoamide dehydrogenase. Alanine racemase and neutral protease were uniquely immunogenic to B. anthracis. Comparative analysis of the spore immunome will be of significance for further nucleic acid- and immuno-based detection systems as well as next-generation vaccine development. PMID:16957262

  7. Serum Proteome Profiling Identifies Novel and Powerful Markers of Cystic Fibrosis Liver Disease

    PubMed Central

    Kügler, Marion; Menendez Menendez, Katrin; Zachoval, Reinhart; Naehrlich, Lutz; Schulz, Richard; Roderfeld, Martin; Roeb, Elke

    2013-01-01

    Background and Aims Cystic Fibrosis associated liver disease (CFLD) develops in approximately 30% of CF patients. However, routine sensitive diagnostic tools for CFLD are lacking. Within this study, we aimed to identify new experimental biomarkers for the detection of CFLD. Methods 45 CF patients were included in the study and received transient elastography. Differential regulation of 220 different serum proteins was assessed in a subgroup of patients with and without CFLD. Most interesting candidate proteins were further quantified and validated by ELISA in the whole patient cohort. To assess a potential relation of biomarker expression to the degree of hepatic fibrosis, serum biomarkers were further determined in 18 HCV patients where liver histology was available. Results 43 serum proteins differed at least 2-fold in patients with CFLD compared to those without liver disease as identified in proteome profiling. In ELISA quantifications, TIMP-4 and Endoglin were significantly up-regulated in patients with CFLD as diagnosed by clinical guidelines or increased liver stiffness. Pentraxin-3 was significantly decreased in patients with CFLD. Serum TIMP-4 and Endoglin showed highest values in HCV patients with liver cirrhosis compared to those with fibrosis but without cirrhosis. At a cut-off value of 6.3 kPa, transient elastography compassed a very high diagnostic accuracy and specificity for the detection of CFLD. Among the biomarkers, TIMP-4 and Endoglin exhibited a high diagnostic accuracy for CFLD. Diagnostic sensitivities and negative predictive values were increased when elastography and TIMP-4 and Endoglin were combined for the detection of CFLD. Conclusions Serum TIMP-4 and Endoglin are increased in CFLD and their expression correlates with hepatic staging. Determination of TIMP-4 and Endoglin together with transient elastography can increase the sensitivity for the non-invasive diagnosis of CFLD. PMID:23516586

  8. Comprehensive analyses of prostate gene expression: convergence of expressed sequence tag databases, transcript profiling and proteomics.

    PubMed

    Nelson, P S; Han, D; Rochon, Y; Corthals, G L; Lin, B; Monson, A; Nguyen, V; Franza, B R; Plymate, S R; Aebersold, R; Hood, L

    2000-05-01

    Several methods have been developed for the comprehensive analysis of gene expression in complex biological systems. Generally these procedures assess either a portion of the cellular transcriptome or a portion of the cellular proteome. Each approach has distinct conceptual and methodological advantages and disadvantages. We have investigated the application of both methods to characterize the gene expression pathway mediated by androgens and the androgen receptor in prostate cancer cells. This pathway is of critical importance for the development and progression of prostate cancer. Of clinical importance, modulation of androgens remains the mainstay of treatment for patients with advanced disease. To facilitate global gene expression studies we have first sought to define the prostate transcriptome by assembling and annotating prostate-derived expressed sequence tags (ESTs). A total of 55000 prostate ESTs were assembled into a set of 15953 clusters putatively representing 15953 distinct transcripts. These clusters were used to construct cDNA microarrays suitable for examining the androgen-response pathway at the level of transcription. The expression of 20 genes was found to be induced by androgens. This cohort included known androgen-regulated genes such as prostate-specific antigen (PSA) and several novel complementary DNAs (cDNAs). Protein expression profiles of androgen-stimulated prostate cancer cells were generated by two-dimensional electrophoresis (2-DE). Mass spectrometric analysis of androgen-regulated proteins in these cells identified the metastasis-suppressor gene NDKA/nm23, a finding that may explain a marked reduction in metastatic potential when these cells express a functional androgen receptor pathway. PMID:10870968

  9. Feeding low or pharmacological concentrations of zinc oxide changes the hepatic proteome profiles in weaned piglets.

    PubMed

    Bondzio, Angelika; Pieper, Robert; Gabler, Christoph; Weise, Christoph; Schulze, Petra; Zentek, Juergen; Einspanier, Ralf

    2013-01-01

    Pharmacological levels of zinc oxide can promote growth and health of weaning piglets, but the underlying molecular mechanisms are yet not fully understood. The aim of this study was to determine changes in the global hepatic protein expression in response to dietary zinc oxide in weaned piglets. Nine half-sib piglets were allocated to three dietary zinc treatment groups (50, 150, 2500 mg/kg dry matter). After 14 d, pigs were euthanized and liver samples taken. The increase in hepatic zinc concentration following dietary supplementation of zinc was accompanied by up-regulation of metallothionein mRNA and protein expression. Global hepatic protein profiles were obtained by two-dimensional difference gel electrophoresis following matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. A total of 15 proteins were differentially (P<0.05) expressed between groups receiving control (150 mg/kg) or pharmacological levels of zinc (2500 mg/kg) with 7 down- (e.g. arginase1, thiosulfate sulfurtransferase, HSP70) and 8 up-regulated (e.g. apolipoprotein AI, transferrin, C1-tetrahydrofolate synthase) proteins. Additionally, three proteins were differentially expressed with low zinc supply (50 mg/kg Zn) in comparison to the control diet. The identified proteins were mainly associated with functions related to cellular stress, transport, metabolism, and signal transduction. The differential regulation was evaluated at the mRNA level and a subset of three proteins of different functional groups was selected for confirmation by western blotting. The results of this proteomic study suggest that zinc affects important liver functions such as blood protein secretion, protein metabolism, detoxification and redox homeostasis, thus supporting the hypothesis of intermediary effects of pharmacological levels of zinc oxide fed to pigs. PMID:24282572

  10. Feeding Low or Pharmacological Concentrations of Zinc Oxide Changes the Hepatic Proteome Profiles in Weaned Piglets

    PubMed Central

    Bondzio, Angelika; Pieper, Robert; Gabler, Christoph; Weise, Christoph; Schulze, Petra; Zentek, Juergen; Einspanier, Ralf

    2013-01-01

    Pharmacological levels of zinc oxide can promote growth and health of weaning piglets, but the underlying molecular mechanisms are yet not fully understood. The aim of this study was to determine changes in the global hepatic protein expression in response to dietary zinc oxide in weaned piglets. Nine half-sib piglets were allocated to three dietary zinc treatment groups (50, 150, 2500 mg/kg dry matter). After 14 d, pigs were euthanized and liver samples taken. The increase in hepatic zinc concentration following dietary supplementation of zinc was accompanied by up-regulation of metallothionein mRNA and protein expression. Global hepatic protein profiles were obtained by two-dimensional difference gel electrophoresis following matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. A total of 15 proteins were differentially (P<0.05) expressed between groups receiving control (150 mg/kg) or pharmacological levels of zinc (2500 mg/kg) with 7 down- (e.g. arginase1, thiosulfate sulfurtransferase, HSP70) and 8 up-regulated (e.g. apolipoprotein AI, transferrin, C1-tetrahydrofolate synthase) proteins. Additionally, three proteins were differentially expressed with low zinc supply (50 mg/kg Zn) in comparison to the control diet. The identified proteins were mainly associated with functions related to cellular stress, transport, metabolism, and signal transduction. The differential regulation was evaluated at the mRNA level and a subset of three proteins of different functional groups was selected for confirmation by western blotting. The results of this proteomic study suggest that zinc affects important liver functions such as blood protein secretion, protein metabolism, detoxification and redox homeostasis, thus supporting the hypothesis of intermediary effects of pharmacological levels of zinc oxide fed to pigs. PMID:24282572

  11. Proteomic and transcriptomic profiling of Staphylococcus aureus surface LPXTG-proteins: correlation with agr genotypes and adherence phenotypes.

    PubMed

    Ythier, Mathilde; Resch, Grégory; Waridel, Patrice; Panchaud, Alexandre; Gfeller, Aurélie; Majcherczyk, Paul; Quadroni, Manfredo; Moreillon, Philippe

    2012-11-01

    Staphylococcus aureus infections involve numerous adhesins and toxins, which expression depends on complex regulatory networks. Adhesins include a family of surface proteins covalently attached to the peptidoglycan via a conserved LPXTG motif. Here we determined the protein and mRNA expression of LPXTG-proteins of S. aureus Newman in time-course experiments, and their relation to fibrinogen adherence in vitro. Experiments were performed with mutants in the global accessory-gene regulator (agr), surface protein A (Spa), and fibrinogen-binding protein A (ClfA), as well as during growth in iron-rich or iron-poor media. Surface proteins were recovered by trypsin-shaving of live bacteria. Released peptides were analyzed by liquid chromatography coupled to tandem mass-spectrometry. To unambiguously identify peptides unique to LPXTG-proteins, the analytical conditions were refined using a reference library of S. aureus LPXTG-proteins heterogeneously expressed in surrogate Lactococcus lactis. Transcriptomes were determined by microarrays. Sixteen of the 18 LPXTG-proteins present in S. aureus Newman were detected by proteomics. Nine LPXTG-proteins showed a bell-shape agr-like expression that was abrogated in agr-negative mutants including Spa, fibronectin-binding protein A (FnBPA), ClfA, iron-binding IsdA, and IsdB, immunomodulator SasH, functionally uncharacterized SasD, biofilm-related SasG and methicillin resistance-related FmtB. However, only Spa and SasH modified their proteomic and mRNA profiles in parallel in the parent and its agr- mutant, whereas all other LPXTG-proteins modified their proteomic profiles independently of their mRNA. Moreover, ClfA became highly transcribed and active in fibrinogen-adherence tests during late growth (24 h), whereas it remained poorly detected by proteomics. On the other hand, iron-regulated IsdA-B-C increased their protein expression by >10-times in iron-poor conditions. Thus, proteomic, transcriptomic, and adherence

  12. [The history and perspective of gender-specific medicine in Japan].

    PubMed

    Shimokawa, Hiroaki

    2015-04-01

    The history of gender-specific medicine started when Barbara Seaman, a female American journalist, started the women's health movement in 1957. Since then, the National Institutes of Health(NIH) and the Food and Drug Administration(FDA) have promoted gender-specific medicine in USA, including the Women's Health Initiative(WHI) by NIH in 1991 and the Office on Women's Health(OWH) by FDA in 1995. In Japan, being stimulated by the movements in USA, Dr. Chuwa Tei founded Japan' s first women' s clinic in Kagoshima University in 2001, and Dr. Keiko Amano founded the Association for Gender-Specific Medicine in 2004. These activities developed into the establishment of the Japanese Association for Gender-Specific Medicine in 2008, for which Dr.Tei served as the first president for 4 years, followed by Hiroaki Shimokawa since 2012. Furthermore, the importance of gender-specific medicine has been emerging worldwide, resulting in the establishment of International Society of Gender Medicine in 2012. Indeed, the history of gender-specific medicine is 60 years worldwide and is only 15 years in Japan. However, gender-specific medicine will become more and more important not only in medicine but also in the whole society. PMID:25936139

  13. PiB-PET Imaging-Based Serum Proteome Profiles Predict Mild Cognitive Impairment and Alzheimer's Disease.

    PubMed

    Kang, Seokjo; Jeong, Hyobin; Baek, Je-Hyun; Lee, Seung-Jin; Han, Sun-Ho; Cho, Hyun Jin; Kim, Hee; Hong, Hyun Seok; Kim, Young Ho; Yi, Eugene C; Seo, Sang Won; Na, Duk L; Hwang, Daehee; Mook-Jung, Inhee

    2016-07-01

    Development of a simple, non-invasive early diagnosis platform of Alzheimer's disease (AD) using blood is urgently required. Recently, PiB-PET imaging has been shown to be powerful to quantify amyloid-β plaque loads leading to pathophysiological alterations in AD brains. Thus, there has been a need for serum biomarkers reflecting PiB-PET imaging data as an early diagnosis platform of AD. Here, using LC-MS/MS analysis coupled with isobaric tagging, we performed comprehensive proteome profiling of serum samples from cognitively normal controls, mild cognitive impairment (MCI), and AD patients, who were selected using PiB-PET imaging. Comparative analysis of the proteomes revealed 79 and 72 differentially expressed proteins in MCI and AD, respectively, compared to controls. Integrated analysis of these proteins with genomic and proteomic data of AD brain tissues, together with network analysis, identified three biomarker candidates representing the altered proteolysis-related process in MCI or AD: proprotein convertase subtilisin/kexin type 9 (PCSK9), coagulation factor XIII, A1 polypeptide (F13A1), and dermcidin (DCD). In independent serum samples of MCI and AD, we confirmed the elevation of the candidates using western blotting and ELISA. Our results suggest that these biomarker candidates can serve as a potential non-invasive early diagnosis platform reflecting PiB-PET imaging for MCI and AD. PMID:27392853

  14. Standardized Profiling of The Membrane-Enriched Proteome of Mouse Dorsal Root Ganglia (DRG) Provides Novel Insights Into Chronic Pain.

    PubMed

    Rouwette, Tom; Sondermann, Julia; Avenali, Luca; Gomez-Varela, David; Schmidt, Manuela

    2016-06-01

    Chronic pain is a complex disease with limited treatment options. Several profiling efforts have been employed with the aim to dissect its molecular underpinnings. However, generated results are often inconsistent and nonoverlapping, which is largely because of inherent technical constraints. Emerging data-independent acquisition (DIA)-mass spectrometry (MS) has the potential to provide unbiased, reproducible and quantitative proteome maps - a prerequisite for standardization among experiments. Here, we designed a DIA-based proteomics workflow to profile changes in the abundance of dorsal root ganglia (DRG) proteins in two mouse models of chronic pain, inflammatory and neuropathic. We generated a DRG-specific spectral library containing 3067 DRG proteins, which enables their standardized quantification by means of DIA-MS in any laboratory. Using this resource, we profiled 2526 DRG proteins in each biological replicate of both chronic pain models and respective controls with unprecedented reproducibility. We detected numerous differentially regulated proteins, the majority of which exhibited pain model-specificity. Our approach recapitulates known biology and discovers dozens of proteins that have not been characterized in the somatosensory system before. Functional validation experiments and analysis of mouse pain behaviors demonstrate that indeed meaningful protein alterations were discovered. These results illustrate how the application of DIA-MS can open new avenues to achieve the long-awaited standardization in the molecular dissection of pathologies of the somatosensory system. Therefore, our findings provide a valuable framework to qualitatively extend our understanding of chronic pain and somatosensation. PMID:27103637

  15. Proteomic profiles of five strains of oxygenic photosynthetic cyanobacteria of the genus Cyanothece.

    PubMed

    Aryal, Uma K; Callister, Stephen J; McMahon, Benjamin H; McCue, Lee-Ann; Brown, Joseph; Stöckel, Jana; Liberton, Michelle; Mishra, Sujata; Zhang, Xiaohui; Nicora, Carrie D; Angel, Thomas E; Koppenaal, David W; Smith, Richard D; Pakrasi, Himadri B; Sherman, Louis A

    2014-07-01

    Members of the cyanobacterial genus Cyanothece exhibit considerable variation in physiological and biochemical characteristics. The comparative assessment of the genomes and the proteomes has the potential to provide insights on differences among Cyanothece strains. By applying Sequedex, an annotation-independent method for ascribing gene functions, we confirmed significant species-specific differences of functional genes in different Cyanothece strains, particularly in Cyanothece PCC7425. Using a shotgun proteomics approach based on prefractionation and tandem mass spectrometry, we detected ∼28-48% of the theoretical Cyanothece proteome, depending on the strain. The expression of a total of 642 orthologous proteins was observed in all five Cyanothece strains. These shared orthologous proteins showed considerable correlations in their abundances across different Cyanothece strains. Functional classification indicated that the majority of proteins involved in central metabolic functions such as amino acid, carbohydrate, protein, and RNA metabolism, photosynthesis, respiration, and stress responses were observed to a greater extent in the core proteome, whereas proteins involved in membrane transport, iron acquisition, regulatory functions, flagellar motility, and chemotaxis were observed to a greater extent in the unique proteome. Considerable differences were evident across different Cyanothece strains. Notably, the analysis of Cyanothece PCC7425, which showed the highest number of unique proteins (682), provided direct evidence of evolutionary differences in this strain. We conclude that Cyanothece PCC7425 diverged significantly from the other Cyanothece strains or evolved from a different lineage. PMID:24846609

  16. Comparative proteomic profiling of cerebrospinal fluid between living and post mortem ALS and control subjects

    PubMed Central

    RANGANATHAN, SRIKANTH; NICHOLL, GEORGINA C.B.; HENRY, SARAH; LUTKA, FRAN; SATHANOORI, RAMASRI; LACOMIS, DAVID; BOWSER, ROBERT

    2010-01-01

    Neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), lack definitive diagnostic tests or biomarkers of disease progression. Most studies that investigate protein abnormalities in ALS have used biofluids such as blood or cerebrospinal fluid (CSF), while some have used post mortem tissue or CSF samples. Since ALS disease progression and post mortem effects probably induce significant alterations to protein modifications or proteolysis, we directly examined the CSF proteome from ALS subjects at various lengths of time from symptom onset and at autopsy by mass spectrometry based proteomics. CSF was also obtained from both healthy age-matched control subjects and at autopsy from healthy and Alzheimer's disease (AD) controls. We identified significant differences in the CSF proteome between living and post mortem ALS subjects, as well as living and post mortem control subjects. We also noted differences in the CSF proteome of ALS subjects that have exhibited symptoms for varying lengths of time and between ALS and AD subjects at end-stage of disease. This is the first study describing differences in the CSF proteome from post mortem and living ALS subjects using a mass spectrometric approach. These differences highlight the importance of utilizing CSF from living ALS subjects near the time of symptom onset for the identification of early protein biomarkers, although some protein alterations that occur early in the disease process are maintained throughout the course of disease and in post mortem samples. PMID:17852009

  17. Proteomics profiling of fiber development and domestication in upland cotton (Gossypium hirsutum L.).

    PubMed

    Hu, Guanjing; Koh, Jin; Yoo, Mi-Jeong; Pathak, Dharminder; Chen, Sixue; Wendel, Jonathan F

    2014-12-01

    Comparative proteomic analyses were performed to detail the evolutionary consequences of strong directional selection for enhanced fiber traits in modern upland cotton (Gossypium hirsutum L.). Using two complementary proteomic approaches, 2-DE and iTRAQ LC-MS/MS, fiber proteomes were examined for four representative stages of fiber development. Approximately 1,000 protein features were characterized using each strategy, collectively resulting in the identification and functional categorization of 1,223 proteins. Unequal contributions of homoeologous proteins were detected for over a third of the fiber proteome, but overall expression was balanced with respect to the genome-of-origin in the allopolyploid G. hirsutum. About 30% of the proteins were differentially expressed during fiber development within wild and domesticated cotton. Notably, domestication was accompanied by a doubling of protein developmental dynamics for the period between 10 and 20 days following pollination. Expression levels of 240 iTRAQ proteins and 293 2-DE spots were altered by domestication, collectively representing multiple cellular and metabolic processes, including metabolism, energy, protein synthesis and destination, defense and stress response. Analyses of homoeolog-specific expression indicate that duplicated gene products in cotton fibers can be differently regulated in response to selection. These results demonstrate the power of proteomics for the analysis of crop domestication and phenotypic evolution. PMID:25156487

  18. Proteomic profiling of Plasmodium falciparum through improved, semiquantitative two-dimensional gel electrophoresis.

    PubMed

    Smit, Salome; Stoychev, Stoyan; Louw, Abraham I; Birkholtz, Lyn-Marie

    2010-05-01

    Two-dimensional gel electrophoresis (2-DE) is one of the most commonly used technologies to obtain a snapshot of the proteome at any specific time. However, its application to study the Plasmodial (malaria parasite) proteome is still limited due to inefficient extraction and detection methods and the extraordinarily large size of some proteins. Here, we report an optimized protein extraction method, the most appropriate methods for Plasmodial protein quantification and 2-DE detection, and finally protein identification by mass spectrometry (MS). Linear detection of Plasmodial proteins in a optimized lysis buffer was only possible with the 2-D Quant kit, and of the four stains investigated, Flamingo Pink was superior regarding sensitivity, linearity, and excellent MS-compatibility. 2-DE analyses of the Plasmodial proteome using this methodology resulted in the reliable detection of 349 spots and a 95% success rate in MS/MS identification. Subsequent application to the analyses of the Plasmodial ring and trophozoite proteomes ultimately resulted in the identification of 125 protein spots, which constituted 57 and 49 proteins from the Plasmodial ring and trophozoite stages, respectively. This study additionally highlights the presence of various isoforms within the Plasmodial proteome, which is of significant biological importance within the Plasmodial parasite during development in the intraerythrocytic developmental cycle. PMID:20218691

  19. Proteomic profiles of five strains of oxygenic photosynthetic cyanobacteria of the genus Cyanothece

    SciTech Connect

    Aryal, Uma K.; Callister, Stephen J.; McMahon, Benjamin H.; McCue, Lee Ann; Brown, Joseph N.; Stockel, Jana; Liberton, Michelle L.; Mishra, Sujata; Zhang, Xiaohui; Nicora, Carrie D.; Angel, Thomas E.; Koppenaal, David W.; Smith, Richard D.; Pakrasi, Himadri B.; Sherman, Louis A.

    2014-07-03

    Members of the cyanobacterial genus Cyanothece exhibit considerable variation in physiological and biochemical characteristics. The comparative assessment of the genomes and the proteomes has the potential to provide insights on differences among Cyanothece strains. By applying Sequedex (http://sequedex.lanl.gov), an annotationindependent method for ascribing gene functions, we confirmed significant speciesspecific differences of functional genes in different Cyanothece strains, particularly in Cyanothece PCC7425. Using a shotgun proteomics approach based on prefractionation and tandem mass spectrometry, we detected ~28-48% of the theoretical Cyanothece proteome depending on the strain. The expression of a total of 642 orthologous proteins was observed in all five Cyanothece strains. These shared orthologous proteins showed considerable correlations in their protein abundances across different Cyanothece strains. Functional classification indicated that the majority of proteins involved in central metabolic functions such as amino acid, carbohydrate, protein and RNA metabolism, photosynthesis, respiration and stress responses were observed to a greater extent in the core proteome, whereas proteins involved in membrane transport, iron acquisition, regulatory functions, flagellar motility and chemotaxis were observed to a greater extent in the unique proteome. Considerable differences were evident across different Cyanothece strains. Notably, the analysis of Cyanothece PCC7425, which showed the highest number of unique proteins (682),

  20. Quantitative proteomics in resected renal cancer tissue for biomarker discovery and profiling

    PubMed Central

    Atrih, A; Mudaliar, M A V; Zakikhani, P; Lamont, D J; Huang, J T-J; Bray, S E; Barton, G; Fleming, S; Nabi, G

    2014-01-01

    Background: Proteomics-based approaches for biomarker discovery are promising strategies used in cancer research. We present state-of-art label-free quantitative proteomics method to assess proteome of renal cell carcinoma (RCC) compared with noncancer renal tissues. Methods: Fresh frozen tissue samples from eight primary RCC lesions and autologous adjacent normal renal tissues were obtained from surgically resected tumour-bearing kidneys. Proteins were extracted by complete solubilisation of tissues using filter-aided sample preparation (FASP) method. Trypsin digested proteins were analysed using quantitative label-free proteomics approach followed by data interpretation and pathways analysis. Results: A total of 1761 proteins were identified and quantified with high confidence (MASCOT ion score threshold of 35 and P-value <0.05). Of these, 596 proteins were identified as differentially expressed between cancer and noncancer tissues. Two upregulated proteins in tumour samples (adipose differentiation-related protein and Coronin 1A) were further validated by immunohistochemistry. Pathway analysis using IPA, KOBAS 2.0, DAVID functional annotation and FLink tools showed enrichment of many cancer-related biological processes and pathways such as oxidative phosphorylation, glycolysis and amino acid synthetic pathways. Conclusions: Our study identified a number of differentially expressed proteins and pathways using label-free proteomics approach in RCC compared with normal tissue samples. Two proteins validated in this study are the focus of on-going research in a large cohort of patients. PMID:24548857

  1. Differential proteomic profile of spermatogenic and Sertoli cells from peri-pubertal testes of three different bovine breeds

    PubMed Central

    Tripathi, Utkarsh K.; Aslam, Muhammad K. M.; Pandey, Shashank; Nayak, Samiksha; Chhillar, Shivani; Srinivasan, A.; Mohanty, T. K.; Kadam, Prashant H.; Chauhan, M. S.; Yadav, Savita; Kumaresan, Arumugam

    2014-01-01

    Sub-fertility is one of the most common problems observed in crossbred males, but the etiology remains unknown in most of the cases. Although proteomic differences in the spermatozoa and seminal plasma between breeds have been investigated, the possible differences at the sperm precursor cells and supporting/nourishing cells have not been studied. The present study reports the differential proteomic profile of spermatogenic and Sertoli cells in crossbred and purebred bulls. Testis was removed by unilateral castration of 12 peri-pubertal bulls (10 months age), four each from crossbred (Holstein Friesian × Tharparkar), exotic purebred [Holstein Friesian (HF)] and indigenous purebred [Tharparkar (TP)] bulls. Spermatogenic and Sertoli cells were isolated and subjected to proteomic analysis. Protein extracts from the Sertoli and spermatogenic cells of each breed were analyzed with 2-dimensional difference gel electrophoresis (2D-DIGE) and analyzed with Decyder™ software. Compared to HF, 26 protein spots were over expressed and 14 protein spots were under expressed in spermatogenic cells of crossbred bulls. Similarly, 7 protein spots were over expressed and 15 protein spots were under expressed in the spermatogenic cells of TP bulls compared to that of crossbred bulls. Out of 12 selected protein spots identified through mass spectrometry, Phosphatidyl ethanolamine binding protein was found to be over expressed in the spermatogenic cells of crossbred bulls compared to TP bulls. The protein, gamma actin was found to be over expressed in the Sertoli cells of HF bulls, whereas Speedy Protein-A was found to be over expressed in Sertoli cells of crossbred bulls. It may be concluded that certain proteomic level differences exist in sperm precursor cells and nourishing cells between breeds, which might be associated with differences in the fertility among these breeds. PMID:25364731

  2. GENDER-SPECIFIC DIFFERENCES IN THE RESPONSE OF MATURING GAMETES TO TOXIC INSULT

    EPA Science Inventory

    GENDER-SPECIFIC DIFFERENCES IN THE RESPONSE OF MATURING GAMETES TO TOXIC INSULT

    Sally D. Perreault, U. S. Environmental Toxicology Division, National Health and Environmental Effects Research Laboratory, Reproductive Toxicology Division, Research Triangle Park, NC 27711

  3. Epigenetic Control of Apolipoprotein E Expression Mediates Gender-Specific Hematopoietic Regulation.

    PubMed

    Vasanthakumar, Aparna; Zullow, Hayley; Lepore, Janet B; Thomas, Kenya; Young, Natalie; Anastasi, John; Reardon, Catherine A; Godley, Lucy A

    2015-12-01

    Epigenetic alterations play a central role in the control of normal and malignant blood cell development. We demonstrate here that expression of a truncated DNA methyltransferase 3B isoform DNMT3B7, which has been shown to alter cellular epigenetic patterns, decreases the overall number of hematopoietic stem and progenitor cells (HSPCs), and markedly diminishes blood cell reconstitution within the female hormonal microenvironment. Gene expression profiling of HSPCs isolated from DNMT3B7 transgenic embryos identified Apolipoprotein E (Apoe) as overexpressed. The CpG island controlling Apoe expression had lower levels of modified cytosines in DNMT3B7 transgenic HSPCs, corresponding with the observed increase in gene expression. Furthermore, we observed that spleens and bone marrows of female mice transplanted with DNMT3B7 transgenic HSPCs express very high levels of Apoe. Finally, the introduction of Apoe-overexpressing HSPCs into male recipients decreased bone marrow engraftment, recapitulating our original observations in female recipients. Our work reveals a dynamic interplay between the intrinsic epigenetic changes in HSPCs and extrinsic endocrine factors acting on these cells to regulate the efficiency of HSPC engraftment and reconstitution. We have identified a novel mechanism by which gender-specific hormones modulate HSPC function, which could serve as a target for augmenting hematopoiesis in cases with limited HSC functionality. PMID:26417967

  4. Proteomic profile of seminal plasma in adolescents and adults with treated and untreated varicocele

    PubMed Central

    Camargo, Mariana; Intasqui, Paula; Bertolla, Ricardo Pimenta

    2016-01-01

    Varicocele, the most important treatable cause of male infertility, is present in 15% of adult males, 35% of men with primary infertility, and 80% of men with secondary infertility. On the other hand, 80% of these men will not present infertility. Therefore, there is a need to differentiate a varicocele that is exerting a deleterious effect that is treatable from a “silent” varicocele. Despite the growing evidence of the cellular effects of varicocele, its underlying molecular mechanisms are still eluding. Proteomics has become a promising area to determine the reproductive biology of semen as well as to improve diagnosis of male infertility. This review aims to discuss the state-of-art in seminal plasma proteomics in patients with varicocele to discuss the challenges in undertaking these studies, as well as the future outlook derived from the growing body of evidence on the seminal proteome. PMID:26643563

  5. Divergent selection for residual feed intake affects the transcriptomic and proteomic profiles of pig skeletal muscle.

    PubMed

    Vincent, A; Louveau, I; Gondret, F; Tréfeu, C; Gilbert, H; Lefaucheur, L

    2015-06-01

    Improving feed efficiency is a relevant strategy to reduce feed cost and environmental waste in livestock production. Selection experiments on residual feed intake (RFI), a measure of feed efficiency, previously indicated that low RFI was associated with lower feed intake, similar growth rate, and greater lean meat content compared with high RFI. To gain insights into the molecular mechanisms underlying these differences, 24 Large White females from 2 lines divergently selected for RFI were examined. Pigs from a low-RFI ("efficient") and high-RFI ("inefficient") line were individually fed ad libitum from 67 d of age (27 kg BW) to slaughter at 115 kg BW (n = 8 per group). Additional pigs of the high-RFI line were feed restricted to the daily feed intake of the ad libitum low-RFI pigs (n = 8) to investigate the impact of selection independently of feed intake. Global gene and protein expression profiles were assessed in the LM collected at slaughter. The analyses involved a porcine commercial microarray and 2-dimensional gel electrophoresis. About 1,000 probes were differentially expressed (P < 0.01) between RFI lines. Only 10% of those probes were also affected by feed restriction. Gene functional classification indicated a greater expression of genes involved in protein synthesis and a lower expression of genes associated with mitochondrial energy metabolism in the low-RFI pigs compared with the high-RFI pigs. At the protein level, 11 unique identified proteins exhibited a differential abundance (P < 0.05) between RFI lines. Differentially expressed proteins were generally not significantly affected by feed restriction. Mitochondrial oxidative proteins such as aconitase hydratase, ATP synthase subunit α, and creatine kinase S-type had a lower abundance in the low-RFI pigs, whereas fructose-biphosphate aldolase A and glyceraldehyde-3-phosphate dehydrogenase, 2 proteins involved in glycolysis, had a greater abundance in those pigs compared with high-RFI pigs

  6. Proteomic profiling: a novel approach to understanding the biological causes of soil water repellency

    NASA Astrophysics Data System (ADS)

    van Keulen, Geertje; Doerr, Stefan H.; Urbanek, Emilia; Jones, Alun; Dudley, Ed

    2010-05-01

    conditions rendering them hydrophilic. The dynamics of production of these proteins and the formation of these hydrophobic protein surfaces in soils are not known. Other, yet unknown, proteins may also contribute to development, reduction and temporal variability of soil water repellency. Here we present the first steps of a new NERC funded project aimed at exploring the relationship between the presence and/or absence of (hydrophobic) protein and soil water repellency. It involves isolation and characterisation of hydrophobic protein and the temporal metaproteomic profiles in UK grassland and dune soils with varying degrees of water repellency. This contributes to identifying the proteomic dynamics, which may influence soil hydrology and structure, and ultimately the ability of soils to absorb water, support biomass growth, store carbon, and to capture and degrade pollutants.

  7. In-depth 2-DE reference map of Aspergillus fumigatus and its proteomic profiling on exposure to itraconazole.

    PubMed

    Gautam, Poonam; Mushahary, Dolly; Hassan, Wazid; Upadhyay, Santosh Kumar; Madan, Taruna; Sirdeshmukh, Ravi; Sundaram, Curam Sreenivasacharlu; Sarma, Puranam Usha

    2016-07-01

    Aspergillus fumigatus (A. fumigatus) is a medically important opportunistic fungus that may lead to invasive aspergillosis in humans with weak immune system. Proteomic profiling of this fungus on exposure to itraconazole (ITC), an azole antifungal drug, may lead to identification of its molecular targets and better understanding on the development of drug resistance against ITC in A. fumigatus. Here, proteome analysis was performed using 2-DE followed by mass spectrometric analysis which resulted in identification of a total of 259 unique proteins. Further, proteome profiling of A. fumigatus was carried out on exposure to ITC, 0.154 μg/ml, the minimum inhibitory concentration (MIC50). Image analysis showed altered levels of 175 proteins (66 upregulated and 109 downregulated) of A. fumigatus treated with ITC as compared to the untreated control. Peptide mass fingerprinting led to the identification of 54 proteins (12 up-regulated and 42 down-regulated). The differentially expressed proteins include proteins related to cell stress, carbohydrate metabolism and amino acid metabolism. We also observed four proteins, including nucleotide phosphate kinase (NDK), that are reported to interact with calcineurin, a protein involved in regulation of cell morphology and fungal virulence. Comparison of differentially expressed proteins on exposure to ITC with artemisinin (ART), an antimalarial drug with antifungal activity(1), revealed a total of 26 proteins to be common among them suggesting that common proteins and pathways are targeted by these two antifungal agents. The proteins targeted by ITC may serve as important leads for development of new antifungal drugs. PMID:26868900

  8. An Integrated Platform for Isolation, Processing, and Mass Spectrometry-based Proteomic Profiling of Rare Cells in Whole Blood*

    PubMed Central

    Li, Siyang; Plouffe, Brian D.; Belov, Arseniy M.; Ray, Somak; Wang, Xianzhe; Murthy, Shashi K.; Karger, Barry L.; Ivanov, Alexander R.

    2015-01-01

    Isolation and molecular characterization of rare cells (e.g. circulating tumor and stem cells) within biological fluids and tissues has significant potential in clinical diagnostics and personalized medicine. The present work describes an integrated platform of sample procurement, preparation, and analysis for deep proteomic profiling of rare cells in blood. Microfluidic magnetophoretic isolation of target cells spiked into 1 ml of blood at the level of 1000–2000 cells/ml, followed by focused acoustics-assisted sample preparation has been coupled with one-dimensional PLOT-LC-MS methodology. The resulting zeptomole detection sensitivity enabled identification of ∼4000 proteins with injection of the equivalent of only 100–200 cells per analysis. The characterization of rare cells in limited volumes of physiological fluids is shown by the isolation and quantitative proteomic profiling of first MCF-7 cells spiked into whole blood as a model system and then two CD133+ endothelial progenitor and hematopoietic cells in whole blood from volunteers. PMID:25755294

  9. Long-term in vivo polychlorinated biphenyl 126 exposure induces oxidative stress and alters proteomic profile on islets of Langerhans

    NASA Astrophysics Data System (ADS)

    Loiola, Rodrigo Azevedo; Dos Anjos, Fabyana Maria; Shimada, Ana Lúcia; Cruz, Wesley Soares; Drewes, Carine Cristiane; Rodrigues, Stephen Fernandes; Cardozo, Karina Helena Morais; Carvalho, Valdemir Melechco; Pinto, Ernani; Farsky, Sandra Helena

    2016-06-01

    It has been recently proposed that exposure to polychlorinated biphenyls (PCBs) is a risk factor to type 2 diabetes mellitus (DM2). We investigated this hypothesis using long-term in vivo PCB126 exposure to rats addressing metabolic, cellular and proteomic parameters. Male Wistar rats were exposed to PCB126 (0.1, 1 or 10 μg/kg of body weight/day; for 15 days) or vehicle by intranasal instillation. Systemic alterations were quantified by body weight, insulin and glucose tolerance, and blood biochemical profile. Pancreatic toxicity was measured by inflammatory parameters, cell viability and cycle, free radical generation, and proteomic profile on islets of Langerhans. In vivo PCB126 exposure enhanced the body weight gain, impaired insulin sensitivity, reduced adipose tissue deposit, and elevated serum triglycerides, cholesterol, and insulin levels. Inflammatory parameters in the pancreas and cell morphology, viability and cycle were not altered in islets of Langerhans. Nevertheless, in vivo PCB126 exposure increased free radical generation and modified the expression of proteins related to oxidative stress on islets of Langerhans, which are indicative of early β-cell failure. Data herein obtained show that long-term in vivo PCB126 exposure through intranasal route induced alterations on islets of Langerhans related to early end points of DM2.

  10. Long-term in vivo polychlorinated biphenyl 126 exposure induces oxidative stress and alters proteomic profile on islets of Langerhans

    PubMed Central

    Loiola, Rodrigo Azevedo; dos Anjos, Fabyana Maria; Shimada, Ana Lúcia; Cruz, Wesley Soares; Drewes, Carine Cristiane; Rodrigues, Stephen Fernandes; Cardozo, Karina Helena Morais; Carvalho, Valdemir Melechco; Pinto, Ernani; Farsky, Sandra Helena

    2016-01-01

    It has been recently proposed that exposure to polychlorinated biphenyls (PCBs) is a risk factor to type 2 diabetes mellitus (DM2). We investigated this hypothesis using long-term in vivo PCB126 exposure to rats addressing metabolic, cellular and proteomic parameters. Male Wistar rats were exposed to PCB126 (0.1, 1 or 10 μg/kg of body weight/day; for 15 days) or vehicle by intranasal instillation. Systemic alterations were quantified by body weight, insulin and glucose tolerance, and blood biochemical profile. Pancreatic toxicity was measured by inflammatory parameters, cell viability and cycle, free radical generation, and proteomic profile on islets of Langerhans. In vivo PCB126 exposure enhanced the body weight gain, impaired insulin sensitivity, reduced adipose tissue deposit, and elevated serum triglycerides, cholesterol, and insulin levels. Inflammatory parameters in the pancreas and cell morphology, viability and cycle were not altered in islets of Langerhans. Nevertheless, in vivo PCB126 exposure increased free radical generation and modified the expression of proteins related to oxidative stress on islets of Langerhans, which are indicative of early β-cell failure. Data herein obtained show that long-term in vivo PCB126 exposure through intranasal route induced alterations on islets of Langerhans related to early end points of DM2. PMID:27292372

  11. Long-term in vivo polychlorinated biphenyl 126 exposure induces oxidative stress and alters proteomic profile on islets of Langerhans.

    PubMed

    Loiola, Rodrigo Azevedo; Dos Anjos, Fabyana Maria; Shimada, Ana Lúcia; Cruz, Wesley Soares; Drewes, Carine Cristiane; Rodrigues, Stephen Fernandes; Cardozo, Karina Helena Morais; Carvalho, Valdemir Melechco; Pinto, Ernani; Farsky, Sandra Helena

    2016-01-01

    It has been recently proposed that exposure to polychlorinated biphenyls (PCBs) is a risk factor to type 2 diabetes mellitus (DM2). We investigated this hypothesis using long-term in vivo PCB126 exposure to rats addressing metabolic, cellular and proteomic parameters. Male Wistar rats were exposed to PCB126 (0.1, 1 or 10 μg/kg of body weight/day; for 15 days) or vehicle by intranasal instillation. Systemic alterations were quantified by body weight, insulin and glucose tolerance, and blood biochemical profile. Pancreatic toxicity was measured by inflammatory parameters, cell viability and cycle, free radical generation, and proteomic profile on islets of Langerhans. In vivo PCB126 exposure enhanced the body weight gain, impaired insulin sensitivity, reduced adipose tissue deposit, and elevated serum triglycerides, cholesterol, and insulin levels. Inflammatory parameters in the pancreas and cell morphology, viability and cycle were not altered in islets of Langerhans. Nevertheless, in vivo PCB126 exposure increased free radical generation and modified the expression of proteins related to oxidative stress on islets of Langerhans, which are indicative of early β-cell failure. Data herein obtained show that long-term in vivo PCB126 exposure through intranasal route induced alterations on islets of Langerhans related to early end points of DM2. PMID:27292372

  12. Skin toxicology of lead species evaluated by their permeability and proteomic profiles: a comparison of organic and inorganic lead.

    PubMed

    Pan, Tai-Long; Wang, Pei-Wen; Al-Suwayeh, Saleh A; Chen, Chih-Chieh; Fang, Jia-You

    2010-08-01

    Lead compounds are known to cause cytotoxicity and genotoxicity. Lead absorption by the skin is an important route through which this metal enters the body. The purpose of this work was to evaluate the skin permeability and toxicological profiles of two lead species, lead acetate and lead nitrate. This study assessed lead-induced toxicity mechanisms by focusing on the histopathology, proteomics, cell growth, and cellular ATP. In vitro skin permeation assays showed that there was no significant difference of lead accumulation within and across the skin between the two lead species. The presence of simulated sweat reduced the skin uptake of lead. The skin deposition of lead acetate was greater than that of lead nitrate with in vivo topical application. On the other hand, lead nitrate produced greater changes in the skin's histology and proteomic profiles compared to lead acetate. Four protein spots which showed significant changes were identified and are discussed in this study. These included glucose-related protein precursor (GRP) 78, K14, alpha-actin, and Rho GDP-dissociation inhibitor 2 (RhoGDI2). These proteins are respectively associated with oxidative stress, apoptosis, wound healing, and proliferation. Lead presented a biphasic pattern on cell growth and intracellular ATP content, with a stimulating effect at low concentrations and an inhibitory effect on cell proliferation at higher concentrations. PMID:20435106

  13. Multiplex Imaging and Cellular Target Identification of Kinase Inhibitors via an Affinity-Based Proteome Profiling Approach

    PubMed Central

    Su, Ying; Pan, Sijun; Li, Zhengqiu; Li, Lin; Wu, Xiaoyuan; Hao, Piliang; Sze, Siu Kwan; Yao, Shao Q.

    2015-01-01

    MLN8237 is a highly potent and presumably selective inhibitor of Aurora kinase A (AKA) and has shown promising antitumor activities. Like other kinase inhibitors which target the ATP-binding site of kinases, MLN8237 might be expected to have potential cellular off-targets. Herein, we report the first photoaffinity-based, small molecule AKA probe capable of both live-cell imaging of AKA activities and in situ proteome profiling of potential off-targets of MLN8237 (including AKA-associating proteins). By using two mutually compatible, bioorthogonal reactions (copper-catalyzed azide-alkyne cycloaddition chemistry and TCO-tetrazine ligation), we demostrate small molecule-based multiplex bioimaging for simultaneous in situ monitoring of two important cell-cycle regulating kinases (AKA and CDK1). A broad range of proteins, as potential off-targets of MLN8237 and AKA's-interacting partners, is subsequently identified by affinity-based proteome profiling coupled with large-scale LC-MS/MS analysis. From these studies, we discover novel AKA interactions which were further validated by cell-based immunoprecipitation (IP) experiments. PMID:25579846

  14. Time-course proteomic profile of Candida albicans during adaptation to a fetal serum.

    PubMed

    Aoki, Wataru; Ueda, Tomomi; Tatsukami, Yohei; Kitahara, Nao; Morisaka, Hironobu; Kuroda, Kouichi; Ueda, Mitsuyoshi

    2013-02-01

    Candida albicans is a commensal organism; however, it causes fatal diseases if the host immunity is compromised. The mortality rate is very high due to the lack of effective treatment, leading to ceaseless demand for novel pharmaceuticals. In this study, time-course proteomics of C. albicans during adaptation to fetal bovine serum (FBS) was described. Time-course proteomics is a promising way to understand the exact process of going adaptation in dynamically changing environments. Candida albicans was cultivated in yeast nitrogen base (YNB) ± FBS media, and we identified 1418 proteins in the endpoint samples incubated for 0 or 60 min by a LC-MS/MS system with a long monolithic silica capillary column. Next, we carried out time-course proteomics of the YNB + FBS samples to identify top-priority proteins for adaption to FBS. We identified 16 proteins as nascent/newly synthesized proteins, and they were recognized as candidates of important virulent factors. Gene ontology analysis revealed that transport-related proteins were enriched in the 16 proteins, indicating that C. albicans probably put priority in time on the acquisition of essential elements. Time-course proteomics of C. albicans revealed the order of priority to adapt to FBS. Depicting time-course dynamics will lead to profound understandings of virulence of C. albicans. PMID:23620121

  15. Integrative proteomic profiling of ovarian cancer cell lines reveals precursor cell associated proteins and functional status

    PubMed Central

    Coscia, F.; Watters, K. M.; Curtis, M.; Eckert, M. A.; Chiang, C. Y.; Tyanova, S.; Montag, A.; Lastra, R. R.; Lengyel, E.; Mann, M.

    2016-01-01

    A cell line representative of human high-grade serous ovarian cancer (HGSOC) should not only resemble its tumour of origin at the molecular level, but also demonstrate functional utility in pre-clinical investigations. Here, we report the integrated proteomic analysis of 26 ovarian cancer cell lines, HGSOC tumours, immortalized ovarian surface epithelial cells and fallopian tube epithelial cells via a single-run mass spectrometric workflow. The in-depth quantification of >10,000 proteins results in three distinct cell line categories: epithelial (group I), clear cell (group II) and mesenchymal (group III). We identify a 67-protein cell line signature, which separates our entire proteomic data set, as well as a confirmatory publicly available CPTAC/TCGA tumour proteome data set, into a predominantly epithelial and mesenchymal HGSOC tumour cluster. This proteomics-based epithelial/mesenchymal stratification of cell lines and human tumours indicates a possible origin of HGSOC either from the fallopian tube or from the ovarian surface epithelium. PMID:27561551

  16. Proteomic Profiling of the Outer Membrane Fraction of the Obligate Intracellular Bacterial Pathogen Ehrlichia ruminantium

    PubMed Central

    Moumène, Amal; Marcelino, Isabel; Ventosa, Miguel; Gros, Olivier; Lefrançois, Thierry; Vachiéry, Nathalie

    2015-01-01

    The outer membrane proteins (OMPs) of Gram-negative bacteria play a crucial role in virulence and pathogenesis. Identification of these proteins represents an important goal for bacterial proteomics, because it aids in vaccine development. Here, we have developed such an approach for Ehrlichia ruminantium, the obligate intracellular bacterium that causes heartwater. A preliminary whole proteome analysis of elementary bodies, the extracellular infectious form of the bacterium, had been performed previously, but information is limited about OMPs in this organism and about their role in the protective immune response. Identification of OMPs is also essential for understanding Ehrlichia’s OM architecture, and how the bacterium interacts with the host cell environment. First, we developed an OMP extraction method using the ionic detergent sarkosyl, which enriched the OM fraction. Second, proteins were separated via one-dimensional electrophoresis, and digested peptides were analyzed via nano-liquid chromatographic separation coupled with mass spectrometry (LC-MALDI-TOF/TOF). Of 46 unique proteins identified in the OM fraction, 18 (39%) were OMPs, including 8 proteins involved in cell structure and biogenesis, 4 in transport/virulence, 1 porin, and 5 proteins of unknown function. These experimental data were compared to the predicted subcellular localization of the entire E. ruminantium proteome, using three different algorithms. This work represents the most complete proteome characterization of the OM fraction in Ehrlichia spp. The study indicates that suitable subcellular fractionation experiments combined with straightforward computational analysis approaches are powerful for determining the predominant subcellular localization of the experimentally observed proteins. We identified proteins potentially involved in E. ruminantium pathogenesis, which are good novel targets for candidate vaccines. Thus, combining bioinformatics and proteomics, we discovered new OMPs

  17. Extending the Limits of Quantitative Proteome Profiling with Data-Independent Acquisition and Application to Acetaminophen-Treated Three-Dimensional Liver Microtissues*

    PubMed Central

    Bruderer, Roland; Bernhardt, Oliver M.; Gandhi, Tejas; Miladinović, Saša M.; Cheng, Lin-Yang; Messner, Simon; Ehrenberger, Tobias; Zanotelli, Vito; Butscheid, Yulia; Escher, Claudia; Vitek, Olga; Rinner, Oliver; Reiter, Lukas

    2015-01-01

    The data-independent acquisition (DIA) approach has recently been introduced as a novel mass spectrometric method that promises to combine the high content aspect of shotgun proteomics with the reproducibility and precision of selected reaction monitoring. Here, we evaluate, whether SWATH-MS type DIA effectively translates into a better protein profiling as compared with the established shotgun proteomics. We implemented a novel DIA method on the widely used Orbitrap platform and used retention-time-normalized (iRT) spectral libraries for targeted data extraction using Spectronaut. We call this combination hyper reaction monitoring (HRM). Using a controlled sample set, we show that HRM outperformed shotgun proteomics both in the number of consistently identified peptides across multiple measurements and quantification of differentially abundant proteins. The reproducibility of HRM in peptide detection was above 98%, resulting in quasi complete data sets compared with 49% of shotgun proteomics. Utilizing HRM, we profiled acetaminophen (APAP)1-treated three-dimensional human liver microtissues. An early onset of relevant proteome changes was revealed at subtoxic doses of APAP. Further, we detected and quantified for the first time human NAPQI-protein adducts that might be relevant for the toxicity of APAP. The adducts were identified on four mitochondrial oxidative stress related proteins (GATM, PARK7, PRDX6, and VDAC2) and two other proteins (ANXA2 and FTCD). Our findings imply that DIA should be the preferred method for quantitative protein profiling. PMID:25724911

  18. Proteomic profiling of an undefined microbial consortium cultured in fermented dairy manure: Methods development.

    PubMed

    Hanson, Andrea J; Paszczynski, Andrzej J; Coats, Erik R

    2016-03-01

    The production of polyhydroxyalkanoates (PHA; bioplastics) from waste or surplus feedstocks using mixed microbial consortia (MMC) and aerobic dynamic feeding (ADF) is a growing field within mixed culture biotechnology. This study aimed to optimize a 2DE workflow to investigate the proteome dynamics of an MMC synthesizing PHA from fermented dairy manure. To mitigate the challenges posed to effective 2DE by this complex sample matrix, the bacterial biomass was purified using Accudenz gradient centrifugation (AGC) before protein extraction. The optimized 2DE method yielded high-quality gels suitable for quantitative comparative analysis and subsequent protein identification by LC-MS/MS. The optimized 2DE method could be adapted to other proteomic investigations involving MMC in complex organic or environmental matrices. PMID:26790989

  19. Two-dimensional fluorescence difference gel electrophoresis for comparative proteomics profiling

    PubMed Central

    Tannu, Nilesh S; Hemby, Scott E

    2007-01-01

    Quantitative proteomics is the workhorse of the modern proteomics initiative. The gel-based and MuDPIT approaches have facilitated vital advances in the measurement of protein expression alterations in normal and disease phenotypic states. The methodological advance in two-dimensional gel electrophoresis (2DGE) has been the multiplexing fluorescent two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). 2D-DIGE is based on direct labeling of lysine groups on proteins with cyanine CyDye DIGE Fluor minimal dyes before isoelectric focusing, enabling the labeling of 2–3 samples with different dyes and electrophoresis of all the samples on the same 2D gel. This capability minimizes spot pattern variability and the number of gels in an experiment while providing simple, accurate and reproducible spot matching. This protocol can be completed in 3–5 weeks depending on the sample size of the experiment and the level of expertise of the investigator. PMID:17487156

  20. Proteomic profiling of bone marrow mesenchymal stem cells upon TGF-beta stimulation

    SciTech Connect

    Wang, Daojing; Park, Jennifer S.; Chu, Julia S.F.; Ari, Krakowski; Luo, Kunxin; Chen, David J.; Li, Song

    2004-08-08

    Bone marrow mesenchymal stem cells (MSCs) can differentiate into different types of cells, and have tremendous potential for cell therapy and tissue engineering. Transforming growth factor {beta}1 (TGF-{beta}) plays an important role in cell differentiation and vascular remodeling. We showed that TGF-{beta} induced cell morphology change and an increase in actin fibers in MSCs. To determine the global effects of TGF-{beta} on MSCs, we employed a proteomic strategy to analyze the effect of TGF-{beta} on the human MSC proteome. By using two-dimensional gel electrophoresis and electrospray ionization coupled to Quadrupole/time-of-flight tandem mass spectrometers, we have generated a proteome reference map of MSCs, and identified {approx}30 proteins with an increase or decrease in expression or phosphorylation in response to TGF-{beta}. The proteins regulated by TGF-{beta} included cytoskeletal proteins, matrix synthesis proteins, membrane proteins, metabolic enzymes, etc. TGF-{beta} increased the expression of smooth muscle (SM) {alpha}-actin and decreased the expression of gelsolin. Over-expression of gelsolin inhibited TGF-{beta}-induced assembly of SM {alpha}-actin; on the other hand, knocking down gelsolin expression enhanced the assembly of {alpha}-actin and actin filaments without significantly affecting {alpha}-actin expression. These results suggest that TGF-{beta} coordinates the increase of {alpha}-actin and the decrease of gelsolin to promote MSC differentiation. This study demonstrates that proteomic tools are valuable in studying stem cell differentiation and elucidating the underlying molecular mechanisms.

  1. SHOTGUN PROTEOMICS: IDENTIFICATION OF UNIQUE PROTEIN PROFILES OF APOPTOTIC BODIES FROM BILIARY EPITHELIAL CELLS

    PubMed Central

    Lleo, Ana; Zhang, Weici; McDonald, W. Hayes; Seeley, Erin H.; Leung, Patrick S.C.; Coppel, Ross L.; Ansari, Aftab A.; Adams, David H.; Afford, Simon; Invernizzi, Pietro; Gershwin, M. Eric

    2014-01-01

    Shotgun proteomics is a powerful analytic method to characterize complex protein mixtures in combination with multi-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). We have used this platform for proteomic characterization of apoptotic bodies in efforts to define the complex protein mixtures found in primary cultures of human intrahepatic biliary epithelial cells (HiBEC), human renal proximal tubular epithelial cells, human bronchial epithelial cells, isolated intrahepatic biliary epithelial cells from explanted primary biliary cirrhosis (PBC) and control liver, using a total of 24 individual samples. Further, as additional controls and for purposes of comparison, proteomic signatures were also obtained from intact cells and apoptotic bodies. The data obtained from LC-MS/MS, combined with database searches and protein assembly algorithms, allowed us to address significant differences in protein spectral counts and identify unique pathways that may be a component to the induction of the signature inflammatory cytokine response against BECs, including the Notch signaling pathway, IL8, IL6, CXCR2 and integrin signaling. Indeed there are 11 proteins that localize specifically to apoptotic bodies of HiBEC and 8 proteins that were specifically absent in HiBEC apoptotic bodies. In conclusion, proteomic analysis of BECs from PBC liver compared to normal liver are significantly different, suggesting that an immunological attack affects the repertoire of proteins expressed and that such cells should be thought of as living in an environment undergoing continuous selection secondary to an innate and adaptive immune response, reflecting an almost “Darwinian” bias. PMID:24841946

  2. Transcriptional and Proteomic Profiling of Aspergillus flavipes in Response to Sulfur Starvation

    PubMed Central

    El-Sayed, Ashraf S. A.; Yassin, Marwa A.; Ali, Gul Shad

    2015-01-01

    Aspergillus flavipes has received considerable interest due to its potential to produce therapeutic enzymes involved in sulfur amino acid metabolism. In natural habitats, A. flavipes survives under sulfur limitations by mobilizing endogenous and exogenous sulfur to operate diverse cellular processes. Sulfur limitation affects virulence and pathogenicity, and modulates proteome of sulfur assimilating enzymes of several fungi. However, there are no previous reports aimed at exploring effects of sulfur limitation on the regulation of A. flavipes sulfur metabolism enzymes at the transcriptional, post-transcriptional and proteomic levels. In this report, we show that sulfur limitation affects morphological and physiological responses of A. flavipes. Transcription and enzymatic activities of several key sulfur metabolism genes, ATP-sulfurylase, sulfite reductase, methionine permease, cysteine synthase, cystathionine β- and γ-lyase, glutathione reductase and glutathione peroxidase were increased under sulfur starvation conditions. A 50 kDa protein band was strongly induced by sulfur starvation, and the proteomic analyses of this protein band using LC-MS/MS revealed similarity to many proteins involved in the sulfur metabolism pathway. PMID:26633307

  3. Proteomic profiling of host-biofilm interactions in an oral infection model resembling the periodontal pocket

    PubMed Central

    Bao, Kai; Belibasakis, Georgios N.; Selevsek, Nathalie; Grossmann, Jonas; Bostanci, Nagihan

    2015-01-01

    Periodontal infections cause inflammatory destruction of the tooth supporting tissues. We recently developed a dynamic, in vitro periodontal organotypic tissue model in a perfusion bioreactor system, in co-culture with an 11-species subgingival biofilm, which may recapitulate early events during the establishment of periodontal infections. This study aimed to characterize the global proteome regulations in this host-biofilm interaction model. Semi-quantitative shotgun proteomics were applied for protein identification and quantification in the co-culture supernatants (human and bacterial) and the biofilm lysates (bacterial). A total of 896 and 3363 proteins were identified as secreted in the supernatant and expressed in the biofilm lysate, respectively. Enriched gene ontology analysis revealed that the regulated secreted human tissue proteins were related to processes of cytoskeletal rearrangement, stress responses, apoptosis, and antigen presentation, all of which are commensurate with deregulated host responses. Most secreted bacterial biofilm proteins derived from their cytoplasmic domain. In the presence of the tissue, the levels of Fusobacterium nucleatum, Actinomyces oris and Campylobacter rectus proteins were significantly regulated. The functions of the up-regulated intracellular (biofilm lysate) proteins were associated with cytokinesis. In conclusion, the proteomic overview of regulated pathways in this host-biofilm interaction model provides insights to the early events of periodontal pathogenesis. PMID:26525412

  4. Proteomic approaches for profiling negative fertility markers in inferior boar spermatozoa

    PubMed Central

    Kwon, Woo-Sung; Oh, Shin-Ae; Kim, Ye-Ji; Rahman, Md Saidur; Park, Yoo-Jin; Pang, Myung-Geol

    2015-01-01

    The ability to predict male fertility is of paramount importance for animal breeding industries and for human reproduction. Conventional semen analysis generally provides information on the quantitative parameters of spermatozoa, but yields no information concerning its functional competence. Proteomics have identified candidates for male fertility biomarkers, but no studies have clearly identified the relationship between the proteome and sperm fertility. Therefore, we performed a proteomic analysis to investigate small and large litter size boar spermatozoa and identify proteins related to male fertility. In this study, 20 proteins showed differential expression levels in small and large litter size groups. Nineteen of these proteins exhibited decreased expression in large litter size samples and increased expression in the small litter group. Interestingly, only one protein was highly expressed in the large litter size spermatozoa. We then identified signaling pathways associated with the differentially expressed protein markers. Glutathione S-transferase Mu3 and glutathione peroxidase 4 were related to the glutathione metabolic pathway and arginine vasopressin receptor 2 was linked to vasopressin R2/STAT. In summary, this is the first study to consider negative fertility biomarkers, and the identified proteins could potentially be used as biomarkers for the detection of inferior male fertility. PMID:26348888

  5. Quantitative Subcellular Proteome and Secretome Profiling of Influenza A Virus-Infected Human Primary Macrophages

    PubMed Central

    Lietzén, Niina; Julkunen, Ilkka; Aittokallio, Tero; Matikainen, Sampsa; Nyman, Tuula A.

    2011-01-01

    Influenza A viruses are important pathogens that cause acute respiratory diseases and annual epidemics in humans. Macrophages recognize influenza A virus infection with their pattern recognition receptors, and are involved in the activation of proper innate immune response. Here, we have used high-throughput subcellular proteomics combined with bioinformatics to provide a global view of host cellular events that are activated in response to influenza A virus infection in human primary macrophages. We show that viral infection regulates the expression and/or subcellular localization of more than one thousand host proteins at early phases of infection. Our data reveals that there are dramatic changes in mitochondrial and nuclear proteomes in response to infection. We show that a rapid cytoplasmic leakage of lysosomal proteins, including cathepsins, followed by their secretion, contributes to inflammasome activation and apoptosis seen in the infected macrophages. Also, our results demonstrate that P2X7 receptor and src tyrosine kinase activity are essential for inflammasome activation during influenza A virus infection. Finally, we show that influenza A virus infection is associated with robust secretion of different danger-associated molecular patterns (DAMPs) suggesting an important role for DAMPs in host response to influenza A virus infection. In conclusion, our high-throughput quantitative proteomics study provides important new insight into host-response against influenza A virus infection in human primary macrophages. PMID:21589892

  6. Global profiling of co- and post-translationally N-myristoylated proteomes in human cells

    PubMed Central

    Thinon, Emmanuelle; Serwa, Remigiusz A.; Broncel, Malgorzata; Brannigan, James A.; Brassat, Ute; Wright, Megan H.; Heal, William P.; Wilkinson, Anthony J.; Mann, David J.; Tate, Edward W.

    2014-01-01

    Protein N-myristoylation is a ubiquitous co- and post-translational modification that has been implicated in the development and progression of a range of human diseases. Here, we report the global N-myristoylated proteome in human cells determined using quantitative chemical proteomics combined with potent and specific human N-myristoyltransferase (NMT) inhibition. Global quantification of N-myristoylation during normal growth or apoptosis allowed the identification of >100 N-myristoylated proteins, >95% of which are identified for the first time at endogenous levels. Furthermore, quantitative dose response for inhibition of N-myristoylation is determined for >70 substrates simultaneously across the proteome. Small-molecule inhibition through a conserved substrate-binding pocket is also demonstrated by solving the crystal structures of inhibitor-bound NMT1 and NMT2. The presented data substantially expand the known repertoire of co- and post-translational N-myristoylation in addition to validating tools for the pharmacological inhibition of NMT in living cells. PMID:25255805

  7. Gender-specific induction of cytochrome P450s in nonylphenol-treated FVB/NJ mice

    SciTech Connect

    Hernandez, Juan P.; Chapman, Laura M.; Kretschmer, Xiomara C.; Baldwin, William S. . E-mail: wbaldwin@utep.edu

    2006-10-15

    Nonylphenol (NP) is a breakdown product of nonylphenol ethoxylates, which are used in a variety of industrial, agricultural, household cleaning, and beauty products. NP is one of the most commonly found toxicants in the United States and Europe and is considered a toxicant of concern because of its long half-life. NP is an environmental estrogen that also activates the pregnane X-receptor (PXR) and in turn induces P450s. No study to date has examined the gender-specific effects of NP on hepatic P450 expression. We provided NP at 0, 50 or 75 mg/kg/day for 7 days to male and female FVB/NJ mice and compared their P450 expression profiles. Q-PCR was performed on hepatic cDNA using primers to several CYP isoforms regulated by PXR or its relative, the constitutive androstane receptor (CAR). In female mice, NP induced Cyp2b10 and Cyp2b13, and downregulated the female-specific P450s, Cyp3a41 and Cyp3a44. In contrast, male mice treated with NP showed increased expression of Cyp2a4, Cyp2b9, and Cyp2b10. Western blots confirmed induction of Cyp2b subfamily members in both males and females. Consistent with the Q-PCR data, Western blots showed dose-dependent downregulation of Cyp3a only in females and induction of Cyp2a only in males. The overall increase in female-predominant P450s in males (Cyp2a4, 2b9) and the decrease in female-predominant P450s in females (Cyp3a41, 3a44) suggest that NP is in part feminizing the P450 profile in males and masculinizing the P450 profile in females. Testosterone hydroxylation was also altered in a gender-specific manner, as testosterone 16{alpha}-hydroxylase activity was only induced in NP-treated males. In contrast, NP-treated females demonstrated a greater propensity for metabolizing zoxazolamine probably due to greater Cyp2b induction in females. In conclusion, NP causes gender-specific P450 induction and therefore exposure to NP may cause distinct pharmacological and toxicological effects in males compared to females.

  8. Differential proteomics profiling of the ova between healthy and Rice stripe virus-infected female insects of Laodelphax striatellus

    PubMed Central

    Liu, Beibei; Qin, Faliang; Liu, Wenwen; Wang, Xifeng

    2016-01-01

    Rice stripe virus-infected females of the small brown planthopper (SBPH, Laodelphax striatellus) usually lay fewer eggs with a longer hatch period, low hatchability, malformation and retarded or defective development compared with healthy females. To explore the molecular mechanism of those phenomena, we analyzed the differential proteomics profiling of the ova between viruliferous and healthy female insects using an isobaric tag for relative and absolute quantitation (iTRAQ) approach. We obtained 147 differentially accumulated proteins: 98 (66.7%) proteins increased, but 49 (33.3%) decreased in the ova of the viruliferous females. RT-qPCR was used to verify the 12 differential expressed proteins from iTRAQ, finding that trends in the transcriptional change for the 12 genes were consistent with those at the proteomic level. Differentially expressed proteins that were associated with meiosis (serine/threonine-protein phosphatase 2B and cyclin B3) and mitosis (cyclin B3 and dynein heavy chain) in viruliferous ova may contribute to low hatchability and defective or retarded development. Alterations in the abundance of proteins involved in the respiratory chain and nutrition metabolism may affect embryonic development. Our study begins to explain macroscopical developmental phenomena and explore the mechanisms by which Rice stripe virus impacts the development of SBPH. PMID:27277140

  9. Plasma proteome profiles of White Sucker (Catostomus commersonii) from the Athabasca River within the oil sands deposit.

    PubMed

    Simmons, Denina B D; Sherry, James P

    2016-09-01

    There are questions about the potential for oil sands related chemicals to enter the Athabasca River, whether from tailing ponds, atmospheric deposition, precipitation, or transport of mining dust, at concentrations sufficient to negatively impact the health of biota. We applied shotgun proteomics to generate protein profiles of mature male and female White Sucker (Catostomus commersonii) that were collected from various sites along the main stem of the Athabasca River in 2011 and 2012. On average, 399±131 (standard deviation) proteins were identified in fish plasma from each location in both years. Ingenuity Pathway Analysis software was used to determine the proteins' core functions and to compare the datasets by location, year, and sex. Principal component analysis (PCA) was used to determine if variation in the number of proteins related to a core function among all male and female individuals from both sampling years was affected by location. The core biological functions of plasma proteins that were common to both sampling years for males and females from each location were also estimated separately (based on Ingenuity's Knowledge Base). PCA revealed site-specific differences in the functional characteristics of the plasma proteome from white sucker sampled from downstream of oil sands extraction facilities compared with fish from upstream. Plasma proteins that were unique to fish downstream of oil sands extraction were related to lipid metabolism, small molecule biochemistry, vitamin and mineral metabolism, endocrine system disorders, skeletal and muscular development and function, neoplasia, carcinomas, and gastrointestinal disease. PMID:27013027

  10. Differential proteomics profiling of the ova between healthy and Rice stripe virus-infected female insects of Laodelphax striatellus.

    PubMed

    Liu, Beibei; Qin, Faliang; Liu, Wenwen; Wang, Xifeng

    2016-01-01

    Rice stripe virus-infected females of the small brown planthopper (SBPH, Laodelphax striatellus) usually lay fewer eggs with a longer hatch period, low hatchability, malformation and retarded or defective development compared with healthy females. To explore the molecular mechanism of those phenomena, we analyzed the differential proteomics profiling of the ova between viruliferous and healthy female insects using an isobaric tag for relative and absolute quantitation (iTRAQ) approach. We obtained 147 differentially accumulated proteins: 98 (66.7%) proteins increased, but 49 (33.3%) decreased in the ova of the viruliferous females. RT-qPCR was used to verify the 12 differential expressed proteins from iTRAQ, finding that trends in the transcriptional change for the 12 genes were consistent with those at the proteomic level. Differentially expressed proteins that were associated with meiosis (serine/threonine-protein phosphatase 2B and cyclin B3) and mitosis (cyclin B3 and dynein heavy chain) in viruliferous ova may contribute to low hatchability and defective or retarded development. Alterations in the abundance of proteins involved in the respiratory chain and nutrition metabolism may affect embryonic development. Our study begins to explain macroscopical developmental phenomena and explore the mechanisms by which Rice stripe virus impacts the development of SBPH. PMID:27277140