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Sample records for reference plasma proteome

  1. Plasma proteome response to severe burn injury revealed by 18O-labeled "universal" reference-based quantitative proteomics.

    PubMed

    Qian, Wei-Jun; Petritis, Brianne O; Kaushal, Amit; Finnerty, Celeste C; Jeschke, Marc G; Monroe, Matthew E; Moore, Ronald J; Schepmoes, Athena A; Xiao, Wenzhong; Moldawer, Lyle L; Davis, Ronald W; Tompkins, Ronald G; Herndon, David N; Camp, David G; Smith, Richard D

    2010-09-03

    A burn injury represents one of the most severe forms of human trauma and is responsible for significant mortality worldwide. Here, we present the first quantitative proteomics investigation of the blood plasma proteome response to severe burn injury by comparing the plasma protein concentrations of 10 healthy control subjects with those of 15 severe burn patients at two time-points following the injury. The overall analytical strategy for this work integrated immunoaffinity depletion of the 12 most abundant plasma proteins with cysteinyl-peptide enrichment-based fractionation prior to LC-MS analyses of individual patient samples. Incorporation of an 18O-labeled "universal" reference among the sample sets enabled precise relative quantification across samples. In total, 313 plasma proteins confidently identified with two or more unique peptides were quantified. Following statistical analysis, 110 proteins exhibited significant abundance changes in response to the burn injury. The observed changes in protein concentrations suggest significant inflammatory and hypermetabolic response to the injury, which is supported by the fact that many of the identified proteins are associated with acute phase response signaling, the complement system, and coagulation system pathways. The regulation of approximately 35 proteins observed in this study is in agreement with previous results reported for inflammatory or burn response, but approximately 50 potentially novel proteins previously not known to be associated with burn response or inflammation are also found. Elucidating proteins involved in the response to severe burn injury may reveal novel targets for therapeutic interventions as well as potential predictive biomarkers for patient outcomes such as multiple organ failure.

  2. Computational clustering for viral reference proteomes.

    PubMed

    Chen, Chuming; Huang, Hongzhan; Mazumder, Raja; Natale, Darren A; McGarvey, Peter B; Zhang, Jian; Polson, Shawn W; Wang, Yuqi; Wu, Cathy H

    2016-07-01

    The enormous number of redundant sequenced genomes has hindered efforts to analyze and functionally annotate proteins. As the taxonomy of viruses is not uniformly defined, viral proteomes pose special challenges in this regard. Grouping viruses based on the similarity of their proteins at proteome scale can normalize against potential taxonomic nomenclature anomalies. We present Viral Reference Proteomes (Viral RPs), which are computed from complete virus proteomes within UniProtKB. Viral RPs based on 95, 75, 55, 35 and 15% co-membership in proteome similarity based clusters are provided. Comparison of our computational Viral RPs with UniProt's curator-selected Reference Proteomes indicates that the two sets are consistent and complementary. Furthermore, each Viral RP represents a cluster of virus proteomes that was consistent with virus or host taxonomy. We provide BLASTP search and FTP download of Viral RP protein sequences, and a browser to facilitate the visualization of Viral RPs. http://proteininformationresource.org/rps/viruses/ chenc@udel.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  3. Temporal profiles of plasma proteome during childhood development.

    PubMed

    Liu, Chih-Wei; Bramer, Lisa; Webb-Robertson, Bobbie-Jo; Waugh, Kathleen; Rewers, Marian J; Zhang, Qibin

    2017-01-30

    Human blood plasma proteome reflects physiological changes associated with a child's development as well as development of disease states. While age-specific normative values are available for proteins routinely measured in clinical practice, there is paucity of comprehensive longitudinal data regarding changes in human plasma proteome during childhood. We applied TMT-10plex isobaric labeling-based quantitative proteomics to longitudinally profile the plasma proteome in 10 healthy children during their development, each with 9 serial time points from 9months to 15years of age. In total, 1828 protein groups were identified at peptide and protein level false discovery rate of 1% and with at least two razor and unique peptides. The longitudinal expression profiles of 1747 protein groups were statistically modeled and their temporal changes were categorized into 7 different patterns. The patterns and relative abundance of proteins obtained by LC-MS were also verified with ELISA. To our knowledge, this study represents the most comprehensive longitudinal profiling of human plasma proteome to date. The temporal profiles of plasma proteome obtained in this study provide a comprehensive resource and reference for biomarker studies in childhood diseases. Biological significance: A pediatric plasma proteome database with longitudinal expression patterns of 1747 proteins from neonate to adolescence was provided to the research community. 970 plasma proteins had age-dependent expression trends, which demonstrated the importance of longitudinal profiling study to identify the potential biomarkers specific to childhood diseases, and the requirement of strictly age-matched clinical samples in a cross-sectional study in pediatric population.

  4. Plasma proteomic profiling of pediatric osteosarcoma.

    PubMed

    Li, Yiting; Dang, Tu Anh; Man, Tsz-Kwong

    2012-01-01

    The development of a sensitive, specific, and non-invasive approach for cancer detection will facilitate early detection and, hence, improve the outcome of individuals with known cancer predispositions. Proteomic profiling of blood emerges to be a logical choice of such non-invasive or minimal invasive detection. However, plasma biomarker discovery of pediatric cancers lags behind that of adult cancers, suggesting more efforts are needed in this area. In this study, we used surface-enhanced laser desorption/ionization-time of flight mass spectrometry to profile plasma proteome in osteosarcoma patients. Osteosarcoma is a bone cancer that affects many children and young adults. We have shown that the plasma proteome contains a unique cancer signature that can distinguish patients with osteosarcoma from those with a benign bone disease. To improve cancer biomarker discovery in plasma, we have also shown that depletion of two highly abundant plasma proteins increases the detection sensitivity of lower-abundance proteins. The combination of depletion and proteomic profiling may increase the chance of identifying tumor-derived proteins within the plasma of pediatric cancer patients.

  5. Optomechanical devices for deep plasma cancer proteomics.

    PubMed

    Kosaka, Priscila M; Calleja, Montserrat; Tamayo, Javier

    2017-09-01

    Most of the cancer deaths could be avoided by early detection of the tumor when it is confined to its primary site and it has not metastasized. To this aim, one of the most promising strategies is the discovery and detection of protein biomarkers shed by the young tumor to the bloodstream. Proteomic technologies, mainly mass spectrometry and multiplexed immunoassays, have rapidly developed during last years with improved limits of detection and multiplexing capability. Unfortunately, these developments together major investments and large international efforts have not resulted into new useful protein biomarkers. Here, we analyze the potential and limitations of current proteomic technologies for detecting protein biomarkers released into circulation by the tumor. We find that these technologies can hardly probe the deepest region of the plasma proteome, at concentrations below the pg/mL level, where protein biomarkers for early cancer detection may exist. This clearly indicates the need of incorporating novel ultrasensitive techniques to the proteomic tool-box that can cover the inaccessible regions of the plasma proteome. We here propose biological detectors based on nanomechanical systems for discovery and detection of cancer protein biomarkers in plasma. We review the modes of operation of these devices, putting our focus on recent developments on nanomechanical sandwich immunoassays and nanomechanical spectrometry. The first technique enables reproducible immunodetection of proteins at concentrations well below the pg/mL level, with a limit of detection on the verge of 10 ag/mL. This technology can potentially detect low abundance tumor-associated proteins in plasma at the very early stages of the tumor. The second technique enables the identification of individual intact proteins by two physical coordinates, the mass and stiffness, instead of the mass-to-charge ratio of the protein constituents. This technology enormously simplifies the identification of

  6. Proteomic analysis of integral plasma membrane proteins.

    PubMed

    Zhao, Yingxin; Zhang, Wei; Kho, Yoonjung; Zhao, Yingming

    2004-04-01

    Efficient methods for profiling proteins integral to the plasma membrane are highly desirable for the identification of overexpressed proteins in disease cells. Such methods will aid in both understanding basic biological processes and discovering protein targets for the design of therapeutic monoclonal antibodies. Avoiding contamination by subcellular organelles and cytosolic proteins is crucial to the successful proteomic analysis of integral plasma membrane proteins. Here we report a biotin-directed affinity purification (BDAP) method for the preparation of integral plasma membrane proteins, which involves (1) biotinylation of cell surface membrane proteins in viable cells, (2) affinity enrichment using streptavidin beads, and (3) depletion of plasma membrane-associated cytosolic proteins by harsh washes with high-salt and high-pH buffers. The integral plasma membrane proteins are then extracted and subjected to SDS-PAGE separation and HPLC/MS/MS for protein identification. We used the BDAP method to prepare integral plasma membrane proteins from a human lung cancer cell line. Western blotting analysis showed that the preparation was almost completely devoid of actin, a major cytosolic protein. Nano-HPLC/MS/MS analysis of only 30 microg of protein extracted from the affinity-enriched integral plasma membrane preparation led to the identification of 898 unique proteins, of which 781 were annotated with regard to their plasma membrane localization. Among the annotated proteins, at least 526 (67.3%) were integral plasma membrane proteins. Notable among them were 62 prenylated proteins and 45 Ras family proteins. To our knowledge, this is the most comprehensive proteomic analysis of integral plasma membrane proteins in mammalian cells to date. Given the importance of integral membrane proteins for drug design, the described approach will expedite the characterization of plasma membrane subproteomes and the discovery of plasma membrane protein drug targets.

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

    PubMed Central

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

    2015-01-01

    Synovial fluid is present in all joint cavities, and protects the articular cartilage surfaces in large by lubricating the joint, thus reducing friction. Several studies have described changes in the protein composition of synovial fluid in patients with joint disease. However, the protein concentration, content, and synovial fluid volume change dramatically during active joint diseases and inflammation, and the proteome composition of healthy synovial fluid is incompletely characterized. We performed a normative proteomics analysis of porcine synovial fluid, and report data from optimizing proteomic methods to investigate the proteome of healthy porcine synovial fluid (Bennike et al., 2014 [1]). We included an evaluation of different proteolytic sample preparation techniques, and an analysis of posttranslational modifications with a focus on glycosylation. We used pig (Sus Scrofa) as a model organism, as the porcine immune system is highly similar to human and the pig genome is sequenced. Furthermore, porcine model systems are commonly used large animal models to study several human diseases. In addition, we analyzed the proteome of human plasma, and compared the proteomes to the obtained porcine synovial fluid proteome. The proteome of the two body fluids were found highly similar, underlining the detected plasma derived nature of many synovial fluid components. The healthy porcine synovial fluid proteomics data, human rheumatoid arthritis synovial fluid proteomics data used in the method optimization, human plasma proteomics data, and search results, have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD000935. PMID:26543887

  8. Comparative Human Salivary and Plasma Proteomes

    PubMed Central

    Loo, J.A.; Yan, W.; Ramachandran, P.; Wong, D.T.

    2010-01-01

    The protein compositions, or the proteomes, found in human salivary and plasma fluids are compared. From recent experimental work by many laboratories, a catalogue of 2290 proteins found in whole saliva has been compiled. This list of salivary proteins is compared with the 2698 proteins found in plasma. Approximately 27% of the whole-saliva proteins are found in plasma. However, despite this apparent low degree of overlap, the distribution found across Gene Ontological categories, such as molecular function, biological processes, and cellular components, shows significant similarities. Moreover, nearly 40% of the proteins that have been suggested to be candidate markers for diseases such as cancer, cardiovascular disease, and stroke can be found in whole saliva. These comparisons and correlations should encourage researchers to consider the use of saliva to discover new protein markers of disease and as a diagnostic non-proximal fluid to detect early signs of disease throughout the body. PMID:20739693

  9. Characterization of the human blood plasma proteome

    SciTech Connect

    Shen, Yufeng; Kim, Jeongkwon; Strittmatter, Eric F.; Jacobs, Jon M.; Camp, David G.; Fang, Ruihua; Tolic, Nikola; Moore, Ronald J.; Smith, Richard D.

    2005-10-15

    We describe methods for broad characterization of the human plasma proteome. The combination of stepwise IgG and albumin protein depletion by affinity chromatography and ultrahigh-efficiency capillary liquid chromatography separations coupled to ion trap-tandem mass spectrometry enabled identification of 2392 proteins from a single plasma sample with an estimated confidence level of >94%, and an additional 2198 proteins with an estimated confidence level of 80%. The relative abundances of the identified proteins span a range of over eight orders of magnitude in concentration (<30 pg/mL to {approx}30 mg/mL), facilitated by the attomole-level sensitivity of the analysis methods. More than 80% of the observed proteins demonstrate interactions with IgG and/or albumin. The results from this study provide a basis for a wide range of plasma proteomics studies, including broad quantitation of relative abundances in comparative studies for the identification of novel protein disease markers, as well as further studies of protein-protein interactions.

  10. Proteomic approaches to identify cold-regulated plasma membrane proteins.

    PubMed

    Takahashi, Daisuke; Nakayama, Takato; Miki, Yushi; Kawamura, Yukio; Uemura, Matsuo

    2014-01-01

    Plasma membrane is the primary determinant of freezing tolerance in plants because of its central role in freeze-thaw cycle. Changes in the plasma membrane proteins have been one of the major research areas in plant cold acclimation. To obtain comprehensive profiles of the plasma membrane proteomes and their changes during the cold acclimation process, a plasma membrane purification method using a dextran-polyethylene glycol two polymer system and a mass spectrometry-based shotgun proteomics method using nano-LC-MS/MS for the plasma membrane proteins are described. The proteomic results obtained are further applied to label-free protein semiquantification.

  11. A reference map of human nasopharyngeal squamous carcinoma proteome.

    PubMed

    Li, Feng; Xiao, Zhiqiang; Zhang, Pengfei; Li, Jianling; Li, Maoyu; Feng, Xueping; Guan, Yongjun; Chen, Zhuchu

    2007-05-01

    In order to conduct a comparative proteomics study of human nasopharyngeal carcinoma (NPC) to understand the molecular mechanisms that participate in the formation of NPC, the two-dimensional gel electrophoresis (2-DE) reference map of human NPC tissue proteome was described. To provide a high level of reproducibility between gels and accurately array each protein expressed in NPC tissue proteome, the two-dimensional polyacrylamide gel electrophoresis system, modified colloidal Coomassie Brilliant Blue staining method and ImageMaster 2D Platinum image analysis software were used. The NPC 2-DE maps show that high quality and good reproducibility of the 2-DE gel pattern was attained. An average total of 1,100 protein spots were separated by 2-DE, visualized by a modified colloidal Coomassie Brilliant Blue staining method. A synthesized 2-DE reference gel was acquired after detailed analysis of the NPC 2-DE gel maps, and 216 medium to high abundant spots were identified as landmark spots of NPC 2-DE gel, which expressed on >75% of gels. To provide an unambiguous identification of the landmark spots in gels, MALDI-TOF, ESI-Q-TOF mass spectrometry and database search were used to identify the proteins expressed in NPC tissue proteome. Between the 216 landmark spots, all proteins were identified with MALDI-TOF at first, 41 of which were identified with both MALDI-TOF and ESI-Q-TOF. All identified proteins were classified in terms of their subcellular localization and physiological function with information from SWISS-PROT and NCBI websites. According to our knowledge this is the first 2-DE reference map of human NPC. This reference map will serve as a basis for further studies of human NPC and the reference map data will be used to expand the proteome database of human NPC, which can be accessed in our website (http://www.xyproteomics.org/).

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

    PubMed

    Geyer, Philipp E; Wewer Albrechtsen, Nicolai J; Tyanova, Stefka; Grassl, Niklas; Iepsen, Eva W; Lundgren, Julie; Madsbad, Sten; Holst, Jens J; Torekov, Signe S; Mann, Matthias

    2016-12-22

    Sustained weight loss is a preferred intervention in a wide range of metabolic conditions, but the effects on an individual's health state remain ill-defined. Here, we investigate the plasma proteomes of a cohort of 43 obese individuals that had undergone 8 weeks of 12% body weight loss followed by a year of weight maintenance. Using mass spectrometry-based plasma proteome profiling, we measured 1,294 plasma proteomes. Longitudinal monitoring of the cohort revealed individual-specific protein levels with wide-ranging effects of losing weight on the plasma proteome reflected in 93 significantly affected proteins. The adipocyte-secreted SERPINF1 and apolipoprotein APOF1 were most significantly regulated with fold changes of -16% and +37%, respectively (P < 10(-13)), and the entire apolipoprotein family showed characteristic differential regulation. Clinical laboratory parameters are reflected in the plasma proteome, and eight plasma proteins correlated better with insulin resistance than the known marker adiponectin. Nearly all study participants benefited from weight loss regarding a ten-protein inflammation panel defined from the proteomics data. We conclude that plasma proteome profiling broadly evaluates and monitors intervention in metabolic diseases. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  13. A Proteomic Study of the HUPO Plasma Proteome Project's Pilot Samples using an Accurate Mass and Time Tag Strategy

    SciTech Connect

    Adkins, Joshua N.; Monroe, Matthew E.; Auberry, Kenneth J.; Shen, Yufeng; Jacobs, Jon M.; Camp, David G.; Vitzthum, Frank; Rodland, Karin D.; Zangar, Richard C.; Smith, Richard D.; Pounds, Joel G.

    2005-08-01

    Characterization of the human blood plasma proteome is critical to the discovery of routinely useful clinical biomarkers. We used an Accurate Mass and Time (AMT) tag strategy with high-resolution mass accuracy capillary liquid chromatography Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry (cLC-FTICR MS) to perform a global proteomic analysis of pilot study samples as part of the HUPO Plasma Proteome Project. HUPO reference serum and citrated plasma samples from African Americans, Asian Americans, and Caucasian Americans were analyzed, in addition to a Pacific Northwest National Laboratory reference serum and plasma. The AMT tag strategy allowed us to leverage two previously published “shotgun” proteomics experiments to perform global analyses on these samples in triplicate in less than 4 days total analysis time. A total of 722 (22% with multiple peptide identifications) International Protein Index (IPI) redundant proteins, or 377 protein families by ProteinProphet, were identified over the 6 individual HUPO serum and plasma samples. The samples yielded a similar number of identified redundant proteins in the plasma samples (average 446 +/-23) as found in the serum samples (average 440+/-20). These proteins were identified by an average of 956+/-35 unique peptides in plasma and 930+/-11 unique peptides in serum. In addition to this high-throughput analysis, the AMT tag approach was used with a Z-score normalization to compare relative protein abundances. This analysis highlighted both known differences in serum and citrated plasma such as fibrinogens, and reproducible differences in peptide abundances from proteins such as soluble activin receptor-like kinase 7b and glycoprotein m6b. The AMT tag strategy not only improved our sample throughput, and provided a basis for estimated quantitation.

  14. Proteomic changes in chicken plasma induced by Salmonella typhimurium lipopolysaccharides

    USDA-ARS?s Scientific Manuscript database

    Lipopolysaccharides (LPS) are cell wall components of gram-negative bacteria that cause inflammation and sickness through genetic and proteomic activation. The objective of our study was to identify the proteomic changes in plasma associated with inflammation induced by LPS treatment. Five-week-old ...

  15. Proteomics applied to transfusion plasma: the beginning of the story.

    PubMed

    Ortiz, A; Richa, L; Defer, C; Dernis, D; Huart, J-J; Tokarski, C; Rolando, C

    2013-05-01

    'Safe blood' is and has always been the major concern in transfusion medicine. Plasma can undergo virus inactivation treatments based on physicochemical, photochemical or thermal methodologies for pathogen inactivation. The validation of these treatments is essentially based on clottability assays and clotting factors' titration; however, their impact on plasma proteins at the molecular level has not yet been evaluated. Proteomics appears as particularly adapted to identify, to localize and, consequently, to correlate these modifications to the biological activity change. At the crossroads of biology and analytical sciences, proteomics is the large-scale study of proteins in tissues, physiological fluids or cells at a given moment and in a precise environment. The proteomic strategy is based on a set of methodologies involving separative techniques like mono- and bidimensional gel electrophoresis and chromatography, analytical techniques, especially mass spectrometry, and bioinformatics. Even if plasma has been extensively studied since the very beginning of proteomics, its application to transfusion medicine has just begun. In the first part of this review, we present the principles of proteomics analysis. Then, we propose a state of the art of proteomics applied to plasma analysis. Finally, the use of proteomics for the evaluation of the impact of storage conditions and pathogen inactivation treatments applied to transfusion plasma and for the evaluation of therapeutic protein fractionated is discussed.

  16. An effective plasma membrane proteomics approach for small tissue samples

    PubMed Central

    Smolders, Katrien; Lombaert, Nathalie; Valkenborg, Dirk; Baggerman, Geert; Arckens, Lutgarde

    2015-01-01

    Advancing the quest for new drug targets demands the development of innovative plasma membrane proteome research strategies applicable to small, functionally defined tissue samples. Biotinylation of acute tissue slices and streptavidin pull-down followed by shotgun proteomics allowed the selective extraction and identification of >1,600 proteins of which >60% are associated with the plasma membrane, including (G-protein coupled) receptors, ion channels and transporters, and this from mm3-scale tissue. PMID:26047021

  17. Identification of new intrinsic proteins in Arabidopsis plasma membrane proteome.

    PubMed

    Marmagne, Anne; Rouet, Marie-Aude; Ferro, Myriam; Rolland, Norbert; Alcon, Carine; Joyard, Jacques; Garin, Jérome; Barbier-Brygoo, Hélène; Ephritikhine, Geneviève

    2004-07-01

    Identification and characterization of anion channel genes in plants represent a goal for a better understanding of their central role in cell signaling, osmoregulation, nutrition, and metabolism. Though channel activities have been well characterized in plasma membrane by electrophysiology, the corresponding molecular entities are little documented. Indeed, the hydrophobic protein equipment of plant plasma membrane still remains largely unknown, though several proteomic approaches have been reported. To identify new putative transport systems, we developed a new proteomic strategy based on mass spectrometry analyses of a plasma membrane fraction enriched in hydrophobic proteins. We produced from Arabidopsis cell suspensions a highly purified plasma membrane fraction and characterized it in detail by immunological and enzymatic tests. Using complementary methods for the extraction of hydrophobic proteins and mass spectrometry analyses on mono-dimensional gels, about 100 proteins have been identified, 95% of which had never been found in previous proteomic studies. The inventory of the plasma membrane proteome generated by this approach contains numerous plasma membrane integral proteins, one-third displaying at least four transmembrane segments. The plasma membrane localization was confirmed for several proteins, therefore validating such proteomic strategy. An in silico analysis shows a correlation between the putative functions of the identified proteins and the expected roles for plasma membrane in transport, signaling, cellular traffic, and metabolism. This analysis also reveals 10 proteins that display structural properties compatible with transport functions and will constitute interesting targets for further functional studies.

  18. Striking against bioterrorism with advanced proteomics and reference methods.

    PubMed

    Armengaud, Jean

    2017-01-01

    The intentional use by terrorists of biological toxins as weapons has been of great concern for many years. Among the numerous toxins produced by plants, animals, algae, fungi, and bacteria, ricin is one of the most scrutinized by the media because it has already been used in biocrimes and acts of bioterrorism. Improving the analytical toolbox of national authorities to monitor these potential bioweapons all at once is of the utmost interest. MS/MS allows their absolute quantitation and exhibits advantageous sensitivity, discriminative power, multiplexing possibilities, and speed. In this issue of Proteomics, Gilquin et al. (Proteomics 2017, 17, 1600357) present a robust multiplex assay to quantify a set of eight toxins in the presence of a complex food matrix. This MS/MS reference method is based on scheduled SRM and high-quality standards consisting of isotopically labeled versions of these toxins. Their results demonstrate robust reliability based on rather loose scheduling of SRM transitions and good sensitivity for the eight toxins, lower than their oral median lethal doses. In the face of an increased threat from terrorism, relevant reference assays based on advanced proteomics and high-quality companion toxin standards are reliable and firm answers.

  19. High Dynamic Range Characterization of the Trauma Patient Plasma Proteome

    SciTech Connect

    Liu, Tao; Qian, Weijun; Gritsenko, Marina A.; Xiao, Wenzhong; Moldawer, Lyle L.; Kaushal, Amit; Monroe, Matthew E.; Varnum, Susan M.; Moore, Ronald J.; Purvine, Samuel O.; Maier, Ronald V.; Davis, Ronald W.; Tompkins, Ronald G.; Camp, David G.; Smith, Richard D.

    2006-06-08

    While human plasma represents an attractive sample for disease biomarker discovery, the extreme complexity and large dynamic range in protein concentrations present significant challenges for characterization, candidate biomarker discovery, and validation. Herein, we describe a strategy that combines immunoaffinity subtraction and chemical fractionation based on cysteinyl peptide and N-glycopeptide captures with 2D-LC-MS/MS to increase the dynamic range of analysis for plasma. Application of this ''divide-and-conquer'' strategy to trauma patient plasma significantly improved the overall dynamic range of detection and resulted in confident identification of 22,267 unique peptides from four different peptide populations (cysteinyl peptides, non-cysteinyl peptides, N-glycopeptides, and non-glycopeptides) that covered 3654 nonredundant proteins. Numerous low-abundance proteins were identified, exemplified by 78 ''classic'' cytokines and cytokine receptors and by 136 human cell differentiation molecules. Additionally, a total of 2910 different N-glycopeptides that correspond to 662 N-glycoproteins and 1553 N-glycosylation sites were identified. A panel of the proteins identified in this study is known to be involved in inflammation and immune responses. This study established an extensive reference protein database for trauma patients, which provides a foundation for future high-throughput quantitative plasma proteomic studies designed to elucidate the mechanisms that underlie systemic inflammatory responses.

  20. High Dynamic Range Characterization of the Trauma Patient Plasma Proteome

    PubMed Central

    Liu, Tao; Qian, Wei-Jun; Gritsenko, Marina A.; Xiao, Wenzhong; Moldawer, Lyle L.; Kaushal, Amit; Monroe, Matthew E.; Varnum, Susan M.; Moore, Ronald J.; Purvine, Samuel O.; Maier, Ronald V.; Davis, Ronald W.; Tompkins, Ronald G.; Camp II, David G.; Smith, Richard D.

    2007-01-01

    SUMMARY While human plasma represents an attractive sample for disease biomarker discovery, the extreme complexity and large dynamic range in protein concentrations present significant challenges for characterization, candidate biomarker discovery, and validation. Herein, we describe a strategy that combines immunoaffinity subtraction and subsequent chemical fractionation based on cysteinyl peptide and N-glycopeptide captures with 2D-LC-MS/MS to increase the dynamic range of analysis for plasma. Application of this “divide-and-conquer” strategy to trauma patient plasma significantly improved the overall dynamic range of detection and resulted in confident identification of 22,267 unique peptides from four different peptide populations (cysteinyl peptides, non-cysteinyl peptides, N-glycopeptides, and non-glycopeptides) that covered 3654 different proteins with 1494 proteins identified by multiple peptides. Numerous low-abundance proteins were identified, exemplified by 78 “classic” cytokines and cytokine receptors and by 136 human cell differentiation molecules. Additionally, a total of 2910 different N-glycopeptides that correspond to 662 N-glycoproteins and 1553 N-glycosylation sites were identified. A panel of the proteins identified in this study is known to be involved in inflammation and immune responses. This study established an extensive reference protein database for trauma patients, which provides a foundation for future high-throughput quantitative plasma proteomic studies designed to elucidate the mechanisms that underlie systemic inflammatory responses. PMID:16684767

  1. Human protein reference database as a discovery resource for proteomics

    PubMed Central

    Peri, Suraj; Navarro, J. Daniel; Kristiansen, Troels Z.; Amanchy, Ramars; Surendranath, Vineeth; Muthusamy, Babylakshmi; Gandhi, T. K. B.; Chandrika, K. N.; Deshpande, Nandan; Suresh, Shubha; Rashmi, B. P.; Shanker, K.; Padma, N.; Niranjan, Vidya; Harsha, H. C.; Talreja, Naveen; Vrushabendra, B. M.; Ramya, M. A.; Yatish, A. J.; Joy, Mary; Shivashankar, H. N.; Kavitha, M. P.; Menezes, Minal; Choudhury, Dipanwita Roy; Ghosh, Neelanjana; Saravana, R.; Chandran, Sreenath; Mohan, Sujatha; Jonnalagadda, Chandra Kiran; Prasad, C. K.; Kumar-Sinha, Chandan; Deshpande, Krishna S.; Pandey, Akhilesh

    2004-01-01

    The rapid pace at which genomic and proteomic data is being generated necessitates the development of tools and resources for managing data that allow integration of information from disparate sources. The Human Protein Reference Database (http://www.hprd.org) is a web-based resource based on open source technologies for protein information about several aspects of human proteins including protein–protein interactions, post-translational modifications, enzyme–substrate relationships and disease associations. This information was derived manually by a critical reading of the published literature by expert biologists and through bioinformatics analyses of the protein sequence. This database will assist in biomedical discoveries by serving as a resource of genomic and proteomic information and providing an integrated view of sequence, structure, function and protein networks in health and disease. PMID:14681466

  2. Low-molecular weight plasma proteome analysis using centrifugal ultrafiltration.

    PubMed

    Greening, David W; Simpson, Richard J

    2011-01-01

    The low-molecular weight fraction (LMF) of the human plasma proteome is an invaluable source of biological information, especially in the context of identifying plasma-based biomarkers of disease. This protocol outlines a standardized procedure for the rapid/reproducible LMF profiling of human plasma samples using centrifugal ultrafiltration fractionation, followed by 1D-SDS-PAGE separation and nano-LC-MS/MS. Ultrafiltration is a convective process that uses anisotropic semipermeable membranes to separate macromolecular species on the basis of size. We have optimized centrifugal ultrafiltration for plasma fractionation with respect to buffer and solvent composition, centrifugal force, duration and temperature to facilitate >95% recovery, and enrichment of low-M (r) components from human plasma. Using this protocol, >260 unique peptides can be identified from a single plasma profiling experiment using 100 μL of plasma (Greening and Simpson, J Proteomics 73:637-648, 2010). The efficacy of this method is demonstrated by the identification, for the first time, of several plasma proteins (e.g., protein KIAA0649 (Q9Y4D3), rheumatoid factor D5, serine protease inhibitor A3, and transmembrane adapter protein PAG) previously not reported in extant high-confidence Human Proteome Organization Plasma Proteome Project datasets.

  3. Liver plasma membranes: an effective method to analyze membrane proteome.

    PubMed

    Cao, Rui; Liang, Songping

    2012-01-01

    Plasma membrane proteins are critical for the maintenance of biological systems and represent important targets for the treatment of disease. The hydrophobicity and low abundance of plasma membrane proteins make them difficult to analyze. The protocols given here are the efficient isolation/digestion procedures for liver plasma membrane proteomic analysis. Both protocol for the isolation of plasma membranes and protocol for the in-gel digestion of gel-embedded plasma membrane proteins are presented. The later method allows the use of a high detergent concentration to achieve efficient solubilization of hydrophobic plasma membrane proteins while avoiding interference with the subsequent LC-MS/MS analysis.

  4. Proteomic profiling of human plasma for cancer biomarker discovery.

    PubMed

    Huang, Zhao; Ma, Linguang; Huang, Canhua; Li, Qifu; Nice, Edouard C

    2017-03-01

    Over the past decades, substantial advances have been made in both the early diagnosis and accurate prognosis of many cancers because of the impressive development of novel proteomic strategies. However, it remains difficult to standardize proteomic approaches. In addition, the heterogeneity of proteins in distinct tissues results in incomplete population of the whole proteome, which inevitably limits its clinical practice. As one of the most complex proteomes in the human body, the plasma proteome contains secreted proteins originating from multiple organs and tissues, making it a favorable matrix for comprehensive biomarker discovery. Here, we will discuss the roles of plasma proteome profiling in cancer biomarker discovery and validation, and highlight both the inherent advantages and disadvantages. Although several hurdles lay ahead, further advances in this technology will greatly increase our understanding of cancer biology, reveal new biomarkers and biomarker panels, and open a new avenue for more efficient early diagnosis and surveillance of cancer, leading toward personalized medicine. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Translational Research and Plasma Proteomic in Cancer.

    PubMed

    Santini, Annamaria Chiara; Giovane, Giancarlo; Auletta, Adelaide; Di Carlo, Angelina; Fiorelli, Alfonso; Cito, Letizia; Astarita, Carlo; Giordano, Antonio; Alfano, Roberto; Feola, Antonia; Di Domenico, Marina

    2016-04-01

    Proteomics is a recent field of research in molecular biology that can help in the fight against cancer through the search for biomarkers that can detect this disease in the early stages of its development. Proteomic is a speedily growing technology, also thanks to the development of even more sensitive and fast mass spectrometry analysis. Although this technique is the most widespread for the discovery of new cancer biomarkers, it still suffers of a poor sensitivity and insufficient reproducibility, essentially due to the tumor heterogeneity. Common technical shortcomings include limitations in the sensitivity of detecting low abundant biomarkers and possible systematic biases in the observed data. Current research attempts are trying to develop high-resolution proteomic instrumentation for high-throughput monitoring of protein changes that occur in cancer. In this review, we describe the basic features of the proteomic tools which have proven to be useful in cancer research, showing their advantages and disadvantages. The application of these proteomic tools could provide early biomarkers detection in various cancer types and could improve the understanding the mechanisms of tumor growth and dissemination.

  6. Plant plasma membrane proteomics for improving cold tolerance.

    PubMed

    Takahashi, Daisuke; Li, Bin; Nakayama, Takato; Kawamura, Yukio; Uemura, Matsuo

    2013-01-01

    Plants are always exposed to various stresses. We have focused on freezing stress, which causes serious problems for agricultural management. When plants suffer freeze-induced damage, the plasma membrane is thought to be the primary site of injury because of its central role in regulation of various cellular processes. Cold tolerant species, however, adapt to such freezing conditions by modifying cellular components and functions (cold acclimation). One of the most important adaptation mechanisms to freezing is alteration of plasma membrane compositions and functions. Advanced proteomic technologies have succeeded in identification of many candidates that may play roles in adaptation of the plasma membrane to freezing stress. Proteomics results suggest that adaptations of plasma membrane functions to low temperature are associated with alterations of protein compositions during cold acclimation. Some of proteins identified by proteomic approaches have been verified their functional roles in freezing tolerance mechanisms further. Thus, accumulation of proteomic results in the plasma membrane is of importance for application to molecular breeding efforts to increase cold tolerance in crops.

  7. A reference map of the Arabidopsis thaliana mature pollen proteome

    SciTech Connect

    Noir, Sandra; Braeutigam, Anne; Colby, Thomas; Schmidt, Juergen; Panstruga, Ralph . E-mail: panstrug@mpiz-koeln.mpg.de

    2005-12-02

    The male gametophyte (or pollen) plays an obligatory role during sexual reproduction of higher plants. The extremely reduced complexity of this organ renders pollen a valuable experimental system for studying fundamental aspects of plant biology such as cell fate determination, cell-cell interactions, cell polarity, and tip-growth. Here, we present the first reference map of the mature pollen proteome of the dicotyledonous model plant species, Arabidopsis thaliana. Based on two-dimensional gel electrophoresis, matrix-assisted laser desorption/ionization time-of-flight, and electrospray quadrupole time-of-flight mass spectrometry, we reproducibly identified 121 different proteins in 145 individual spots. The presence, subcellular localization, and functional classification of the identified proteins are discussed in relation to the pollen transcriptome and the full protein complement encoded by the nuclear Arabidopsis genome.

  8. The human plasma proteome: history, character, and diagnostic prospects.

    PubMed

    Anderson, N Leigh; Anderson, Norman G

    2002-11-01

    The human plasma proteome holds the promise of a revolution in disease diagnosis and therapeutic monitoring provided that major challenges in proteomics and related disciplines can be addressed. Plasma is not only the primary clinical specimen but also represents the largest and deepest version of the human proteome present in any sample: in addition to the classical "plasma proteins," it contains all tissue proteins (as leakage markers) plus very numerous distinct immunoglobulin sequences, and it has an extraordinary dynamic range in that more than 10 orders of magnitude in concentration separate albumin and the rarest proteins now measured clinically. Although the restricted dynamic range of conventional proteomic technology (two-dimensional gels and mass spectrometry) has limited its contribution to the list of 289 proteins (tabulated here) that have been reported in plasma to date, very recent advances in multidimensional survey techniques promise at least double this number in the near future. Abundant scientific evidence, from proteomics and other disciplines, suggests that among these are proteins whose abundances and structures change in ways indicative of many, if not most, human diseases. Nevertheless, only a handful of proteins are currently used in routine clinical diagnosis, and the rate of introduction of new protein tests approved by the United States Food and Drug Administration (FDA) has paradoxically declined over the last decade to less than one new protein diagnostic marker per year. We speculate on the reasons behind this large discrepancy between the expectations arising from proteomics and the realities of clinical diagnostics and suggest approaches by which protein-disease associations may be more effectively translated into diagnostic tools in the future.

  9. Statistical Analysis of Variation in the Human Plasma Proteome

    DOE PAGES

    Corzett, Todd H.; Fodor, Imola K.; Choi, Megan W.; ...

    2010-01-01

    Quantifying the variation in the human plasma proteome is an essential prerequisite for disease-specific biomarker detection. We report here on the longitudinal and individual variation in human plasma characterized by two-dimensional difference gel electrophoresis (2-D DIGE) using plasma samples from eleven healthy subjects collected three times over a two week period. Fixed-effects modeling was used to remove dye and gel variability. Mixed-effects modeling was then used to quantitate the sources of proteomic variation. The subject-to-subject variation represented the largest variance component, while the time-within-subject variation was comparable to the experimental variation found in a previous technical variability study where onemore » human plasma sample was processed eight times in parallel and each was then analyzed by 2-D DIGE in triplicate. Here, 21 protein spots had larger than 50% CV, suggesting that these proteins may not be appropriate as biomarkers and should be carefully scrutinized in future studies. Seventy-eight protein spots showing differential protein levels between different individuals or individual collections were identified by mass spectrometry and further characterized using hierarchical clustering. The results present a first step toward understanding the complexity of longitudinal and individual variation in the human plasma proteome, and provide a baseline for improved biomarker discovery.« less

  10. HUPO Plasma Proteome Project specimen collection and handling: towards the standardization of parameters for plasma proteome samples.

    PubMed

    Rai, Alex J; Gelfand, Craig A; Haywood, Bruce C; Warunek, David J; Yi, Jizu; Schuchard, Mark D; Mehigh, Richard J; Cockrill, Steven L; Scott, Graham B I; Tammen, Harald; Schulz-Knappe, Peter; Speicher, David W; Vitzthum, Frank; Haab, Brian B; Siest, Gerard; Chan, Daniel W

    2005-08-01

    There is a substantial list of pre-analytical variables that can alter the analysis of blood-derived samples. We have undertaken studies on some of these issues including choice of sample type, stability during storage, use of protease inhibitors, and clinical standardization. As there is a wide range of sample variables and a broad spectrum of analytical techniques in the HUPO PPP effort, it is not possible to define a single list of pre-analytical standards for samples or their processing. We present here a compendium of observations, drawing on actual results and sound clinical theories and practices. Based on our data, we find that (1) platelet-depleted plasma is preferable to serum for certain peptidomic studies; (2) samples should be aliquoted and stored preferably in liquid nitrogen; (3) the addition of protease inhibitors is recommended, but should be incorporated early and used judiciously, as some form non specific protein adducts and others interfere with peptide studies. Further, (4) the diligent tracking of pre-analytical variables and (5) the use of reference materials for quality control and quality assurance, are recommended. These findings help provide guidance on sample handling issues, with the overall suggestion being to be conscious of all possible pre-analytical variables as a prerequisite of any proteomic study.

  11. Plasma proteomics, the Human Proteome Project, and cancer-associated alternative splice variant proteins.

    PubMed

    Omenn, Gilbert S

    2014-05-01

    This article addresses three inter-related subjects: the development of the Human Plasma Proteome Peptide Atlas, the launch of the Human Proteome Project, and the emergence of alternative splice variant transcripts and proteins as important features of evolution and pathogenesis. The current Plasma Peptide Atlas provides evidence on which peptides have been detected for every protein confidently identified in plasma; there are links to their spectra and their estimated abundance, facilitating the planning of targeted proteomics for biomarker studies. The Human Proteome Project (HPP) combines a chromosome-centric C-HPP with a biology and disease-driven B/D-HPP, upon a foundation of mass spectrometry, antibody, and knowledgebase resource pillars. The HPP aims to identify the approximately 7000 "missing proteins" and to characterize all proteins and their many isoforms. Success will enable the larger research community to utilize newly-available peptides, spectra, informative MS transitions, and databases for targeted analyses of priority proteins for each organ and disease. Among the isoforms of proteins, splice variants have the special feature of greatly enlarging protein diversity without enlarging the genome; evidence is accumulating of striking differential expression of splice variants in cancers. In this era of RNA-sequencing and advanced mass spectrometry, it is no longer sufficient to speak simply of increased or decreased expression of genes or proteins without carefully examining the splice variants in the protein mixture produced from each multi-exon gene. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge. Copyright © 2013. Published by Elsevier B.V.

  12. [Experimental estimation of proteome size for cells and human plasma].

    PubMed

    Naryzhny, S N; Zgoda, V G; Maynskova, M A; Ronzhina, N L; Belyakova, N V; Legina, O K; Archakov, A I

    2015-01-01

    Huge range of concentrations of different protein and insufficient sensitivity of methods for detection of proteins at a single molecule level does not yet allow obtaining the whole image of human proteome. In our investigations, we tried to evaluate the size of different proteomes (cells and plasma). The approach used is based on detection of protein spots in 2-DE after staining by protein dyes with different sensitivities. The function representing the dependence of the number of protein spots on sensitivity of protein dyes was generated. Next, by extrapolation of this function curve to theoretical point of the maximum sensitivity (detection of a single smallest polypeptide) it was calculated that a single human cell (HepG2) may contain minimum 70,000 proteoforms, and plasma--1.5 mln. Utilization of this approach to other, smaller proteomes showed the competency of this extrapolation. For instance, the size of mycoplas ma (Acholeplasma laidlawii) was estimated in 1100 proteoforms, yeast (Saccharomyces cerevisiae)--40,000, E. coli--6200, P. furiosus--3400. In hepatocytes, the amount of proteoforms was the same as in HepG2--70,000. Significance of obtained data is in possibilities to estimating the proteome organization and planning next steps in its study.

  13. Proteomics of Microparticles with SILAC Quantification (PROMIS-Quan): A Novel Proteomic Method for Plasma Biomarker Quantification*

    PubMed Central

    Harel, Michal; Oren-Giladi, Pazit; Kaidar-Person, Orit; Shaked, Yuval; Geiger, Tamar

    2015-01-01

    Unbiased proteomic analysis of plasma samples holds the promise to reveal clinically invaluable disease biomarkers. However, the tremendous dynamic range of the plasma proteome has so far hampered the identification of such low abundant markers. To overcome this challenge we analyzed the plasma microparticle proteome, and reached an unprecedented depth of over 3000 plasma proteins in single runs. To add a quantitative dimension, we developed PROMIS-Quan—PROteomics of MIcroparticles with Super-Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) Quantification, a novel mass spectrometry-based technology for plasma microparticle proteome quantification. PROMIS-Quan enables a two-step relative and absolute SILAC quantification. First, plasma microparticle proteomes are quantified relative to a super-SILAC mix composed of cell lines from distinct origins. Next, the absolute amounts of selected proteins of interest are quantified relative to the super-SILAC mix. We applied PROMIS-Quan to prostate cancer and compared plasma microparticle samples of healthy individuals and prostate cancer patients. We identified in total 5374 plasma-microparticle proteins, and revealed a predictive signature of three proteins that were elevated in the patient-derived plasma microparticles. Finally, PROMIS-Quan enabled determination of the absolute quantitative changes in prostate specific antigen (PSA) upon treatment. We propose PROMIS-Quan as an innovative platform for biomarker discovery, validation, and quantification in both the biomedical research and in the clinical worlds. PMID:25624350

  14. Proteomic identification of turkey (Meleagris gallopavo) seminal plasma proteins.

    PubMed

    Slowinska, M; Nynca, J; Arnold, G J; Fröhlich, T; Jankowski, J; Kozlowski, K; Mostek, A; Ciereszko, A

    2017-09-01

    SDS-PAGE combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and 2-dimensional electrophoresis (2DE) combined with matrix-assisted laser desorption/ionization time of flight/time of flight mass spectrometry (MALDI TOF/TOF) were applied to characterize the turkey seminal plasma proteome. LC-MS/MS led to the identification of 175 proteins, which were classified according to their function and to corresponding biochemical pathways. Using 2DE and MALDI TOF/TOF, 34 different turkey seminal plasma proteins could be identified, of which 20 were found in more than one spot, indicating different proteoforms of these proteins. For validation, antibodies against turkey albumin and ovoinhibitor as well as sperm acrosin were used in 2DE Western blots experiments. The bioinformatic analysis of the results indicates that turkey seminal plasma proteins may be involved in regulation of lipid metabolism [liver X receptor/retinoid X receptor (LXR/RXR) activation and farnesoid X receptor/retinoid X receptor (FXR/RXR) activation pathways)], endocytic entry of proteins and lipids at the plasma membrane (clathrin-mediated endocytosis pathway), and defense against pathogens (acute phase response signaling pathway) and energy production (glycolysis and gluconeogenesis). Moreover, a comparative meta-analysis of seminal plasma proteomes from other species indicated the presence of proteins specific for avian reproduction, but distinct differences between turkey and chicken seminal plasma proteomes were detected. The results of our study provide basic knowledge of the protein composition of turkey seminal plasma highlighting important physiological pathways which may play crucial roles in the sperm environment after ejaculation. This knowledge can be the basis to further develop procedures improving the reproduction of farmed turkeys. © 2017 Poultry Science Association Inc.

  15. Association between the seminal plasma proteome and sperm functional traits.

    PubMed

    Intasqui, Paula; Camargo, Mariana; Antoniassi, Mariana Pereira; Cedenho, Agnaldo Pereira; Carvalho, Valdemir Melechco; Cardozo, Karina Helena Morais; Zylbersztejn, Daniel Suslik; Bertolla, Ricardo Pimenta

    2016-03-01

    To analyze the seminal plasma proteome and biological functions associated with sperm functional alterations. Cross-sectional study. University andrology and research laboratories. A total of 156 normozoospermic men. Sperm mitochondrial activity, acrosome integrity, and DNA fragmentation were evaluated in a semen aliquot. Remaining semen was centrifuged, and seminal plasma was utilized for proteomic analysis (liquid chromatography-tandem mass spectrometry). Patients were divided into percentiles (15%) to form the following groups: substudy 1, high (control, n = 26) and low (study, n = 23) sperm mitochondrial activity; substudy 2, high (control, n = 23) and low (study, n = 22) sperm acrosome integrity; and substudy 3, low (control, n = 22) and high (study, n = 22) sperm DNA fragmentation. Groups were compared using univariate and multivariate analyses. Differentially expressed proteins were used for functional enrichment analysis. Seminal plasma proteome and postgenomic pathways are associated with several sperm functional traits. In total, 506, 493, and 464 proteins were observed in substudies 1, 2, and 3, respectively. Enriched functions in substudy 1 were intramolecular oxidoreductase activity, aminoglycans catabolism, endopeptidases inhibition, lysosomes, and acute-phase response (study group). In substudy 2, main enriched functions were phospholipase inhibition, arachidonic acid metabolism, exocytosis, regulation of acute inflammation, response to hydrogen peroxide, and lysosomal transport (study group). In substudy 3, enriched functions were prostaglandin biosynthesis and fatty acid binding (study group). We proposed eight, six, and eight seminal biomarkers for substudies 1, 2, and 3, respectively. Seminal plasma proteome reflects sperm mitochondrial activity reduction, acrosome damage, and DNA fragmentation, with several postgenomic functions related to these alterations. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc

  16. Method and platform standardization in MRM-based quantitative plasma proteomics.

    PubMed

    Percy, Andrew J; Chambers, Andrew G; Yang, Juncong; Jackson, Angela M; Domanski, Dominik; Burkhart, Julia; Sickmann, Albert; Borchers, Christoph H

    2013-12-16

    There exists a growing demand in the proteomics community to standardize experimental methods and liquid chromatography-mass spectrometry (LC/MS) platforms in order to enable the acquisition of more precise and accurate quantitative data. This necessity is heightened by the evolving trend of verifying and validating candidate disease biomarkers in complex biofluids, such as blood plasma, through targeted multiple reaction monitoring (MRM)-based approaches with stable isotope-labeled standards (SIS). Considering the lack of performance standards for quantitative plasma proteomics, we previously developed two reference kits to evaluate the MRM with SIS peptide approach using undepleted and non-enriched human plasma. The first kit tests the effectiveness of the LC/MRM-MS platform (kit #1), while the second evaluates the performance of an entire analytical workflow (kit #2). Here, these kits have been refined for practical use and then evaluated through intra- and inter-laboratory testing on 6 common LC/MS platforms. For an identical panel of 22 plasma proteins, similar concentrations were determined, regardless of the kit, instrument platform, and laboratory of analysis. These results demonstrate the value of the kit and reinforce the utility of standardized methods and protocols. The proteomics community needs standardized experimental protocols and quality control methods in order to improve the reproducibility of MS-based quantitative data. This need is heightened by the evolving trend for MRM-based validation of proposed disease biomarkers in complex biofluids such as blood plasma. We have developed two kits to assist in the inter- and intra-laboratory quality control of MRM experiments: the first kit tests the effectiveness of the LC/MRM-MS platform (kit #1), while the second evaluates the performance of an entire analytical workflow (kit #2). In this paper, we report the use of these kits in intra- and inter-laboratory testing on 6 common LC/MS platforms. This

  17. Utilizing human blood plasma for proteomic biomarker discovery

    SciTech Connect

    Jacobs, Jon M.; Adkins, Joshua N.; Qian, Weijun; Liu, Tao; Shen, Yufeng; Camp, David G.; Smith, Richard D.

    2005-08-01

    Application of proteomic biomarker discovery efforts towards human plasma entails both incredible clinical potential as well as significant challenges to overcome the intrinsic characteristics of plasma. The dynamic range of proteins within plasma, coupled with the likely presence of potential biomarkers in the more difficult to detect lower abundance range has driven the development of various methodologies and strategies to maximize the possible detective dynamic range within this biofluid. Discussed is the array of the available approaches currently used by our laboratory and others to utilized human plasma as a viable medium for biomarker discovery efforts. Various separation, depletion, enrichment, and quantitative efforts have resulted in a measurable improvement in the detectability of the low abundance fraction of proteins but more advances are needed to bridge the gap between the current range of detection and what remains unobservable to fully maximize the potential of this sample.

  18. A proteome reference map of the causative agent of melioidosis Burkholderia pseudomallei.

    PubMed

    Wongtrakoongate, Patompon; Roytrakul, Sittiruk; Yasothornsrikul, Sukkid; Tungpradabkul, Sumalee

    2011-01-01

    Burkholderia pseudomallei is the etiologic agent of melioidosis. Using 2DE and MALDI-TOF MS, we report here a proteome reference map constructed from early stationary phase, a bacterial adaptation process. We identified 282 protein spots representing 220 ORFs; many of them have been implicated in bacterial pathogenesis. Up to 20% of identified ORFs belong to post-translational modification and stress responses. The proteome reference map will support future analysis of the bacterial gene and environmental regulation and facilitate comparative proteomics with its sibling species.

  19. Data management and data integration in the HUPO plasma proteome project.

    PubMed

    Omenn, Gilbert S

    2011-01-01

    The Human Plasma Proteome Project (HPPP) is an international collaboration coordinated by the Human Proteome Organisation (HUPO). Its Pilot Phase generated the 2005 Proteomics special issue "Exploring the Human Plasma Proteome" (Omenn et al. Proteomics 5:3226-3245, 2005) and a book with the same title (Omenn GS (ed) (2006) Exploring the human plasma proteome. Wiley-Liss, Weinheim, pp 372). Data management for that Pilot Phase included collection, integration, analysis, and dissemination of findings from participating laboratories and data repositories. Many investigators face the same challenges of integration of data from complex, dynamic serum, and plasma specimens. The PPP workflow assembled a representative Core Dataset of 3,020 protein identifications, overcoming ambiguity and redundancy in the heterogeneous contributed identifications and redundancy and updates in the protein sequence databases. The results were made available with alternative thresholds from the University of Michigan, yielding a range of numbers of protein identifications. Data were submitted to EBI/PRIDE and to ISB/PeptideAtlas. The current phase of the PPP employs Proteome Xchange to link submission of well-annotated primary datasets to EBI/PRIDE, distributed file sharing by Tranche/Proteome Commons.org, and reanalysis from the primary raw spectra at ISB/PeptideAtlas. Such human plasma proteome datasets are available for data mining comparisons with the proteomes of other organs and biofluids in health and disease.

  20. Plasma chemistry references values in psittaciformes.

    PubMed

    Lumeij, J T; Overduin, L M

    1990-04-01

    Reference values for 17 plasma chemical variables in African greys. Amazons, cockatoos and macaws were established for use in avian clinical practice. The inner limits are given for the percentiles P(2.5) and P(97.5) with a probability of 90%. The following variables were studied: urea, creatinine, uric acid, urea/uric acid ratio, osmolality, sodium, potassium, calcium, glucose, aspartate aminotransferase, alanine aminotransferase, gamma glutamyltransferase, lactate dehydrogenase, creatine kinase, bile acids, total protein, albumin/globulin ratio. Differences between methods used and values found in this study and those reported previously are discussed.

  1. 18O-labeled proteome reference as global internal standards for targeted quantification by selected reaction monitoring-mass spectrometry.

    PubMed

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

    2011-12-01

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

  2. 18O-Labeled Proteome Reference as Global Internal Standards for Targeted Quantification by Selected Reaction Monitoring-Mass Spectrometry*

    PubMed Central

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

    2011-01-01

    Selected reaction monitoring (SRM)-MS is an emerging technology for high throughput targeted protein quantification and verification in biomarker discovery studies; however, the cost associated with the application of stable isotope-labeled synthetic peptides as internal standards can be prohibitive for screening a large number of candidate proteins as often required in the preverification phase of discovery studies. Herein we present a proof of concept study using an 18O-labeled proteome reference as global internal standards (GIS) for SRM-based relative quantification. The 18O-labeled proteome reference (or GIS) can be readily prepared and contains a heavy isotope (18O)-labeled internal standard for every possible tryptic peptide. Our results showed that the percentage of heavy isotope (18O) incorporation applying an improved protocol was >99.5% for most peptides investigated. The accuracy, reproducibility, and linear dynamic range of quantification were further assessed based on known ratios of standard proteins spiked into the labeled mouse plasma reference. Reliable quantification was observed with high reproducibility (i.e. coefficient of variance <10%) for analyte concentrations that were set at 100-fold higher or lower than those of the GIS based on the light (16O)/heavy (18O) peak area ratios. The utility of 18O-labeled GIS was further illustrated by accurate relative quantification of 45 major human plasma proteins. Moreover, quantification of the concentrations of C-reactive protein and prostate-specific antigen was illustrated by coupling the GIS with standard additions of purified protein standards. Collectively, our results demonstrated that the use of 18O-labeled proteome reference as GIS provides a convenient, low cost, and effective strategy for relative quantification of a large number of candidate proteins in biological or clinical samples using SRM. PMID:21988777

  3. Site specific modification of the human plasma proteome by methylglyoxal.

    PubMed

    Kimzey, Michael J; Kinsky, Owen R; Yassine, Hussein N; Tsaprailis, George; Stump, Craig S; Monks, Terrence J; Lau, Serrine S

    2015-12-01

    Increasing evidence identifies dicarbonyl stress from reactive glucose metabolites, such as methylglyoxal (MG), as a major pathogenic link between hyperglycemia and complications of diabetes. MG covalently modifies arginine residues, yet the site specificity of this modification has not been thoroughly investigated. Sites of MG adduction in the plasma proteome were identified using LC-MS/MS analysis in vitro following incubation of plasma proteins with MG. Treatment of plasma proteins with MG yielded 14 putative MG hotspots from five plasma proteins (albumin [nine hotspots], serotransferrin, haptoglobin [2 hotspots], hemopexin, and Ig lambda-2 chain C regions). The search results revealed two versions of MG-arginine modification, dihydroxyimidazolidine (R+72) and hydroimidazolone (R+54) adducts. One of the sites identified was R257 in human serum albumin, which is a critical residue located in drug binding site I. This site was validated as a target for MG modification by a fluorescent probe displacement assay, which revealed significant drug dissociation at 300 μM MG from a prodan-HSA complex (75 μM). Moreover, twelve human plasma samples (six male, six female, with two type 2 diabetic subjects from both genders) were analyzed using multiple reaction monitoring (MRM) tandem mass spectrometry and revealed the presence of the MG-modified albumin R257 peptide. These data provide insights into the nature of the site-specificity of MG modification of arginine, which may be useful for therapeutic treatments that aim to prevent MG-mediated adverse responses in patients.

  4. Data Management and Data Integration in the HUPO Plasma Proteome Project

    PubMed Central

    Omenn, Gilbert S.

    2017-01-01

    The Human Plasma Proteome Project (HPPP) is an international collaboration coordinated by the Human Proteome Organisation (HUPO). Its Pilot Phase generated the 2005 Proteomics special issue “Exploring the Human Plasma Proteome” (Omenn et al. Proteomics 5:3226–3245, 2005) and a book with the same title (Omenn GS (ed) (2006) Exploring the human plasma proteome. Wiley-Liss, Weinheim, pp 372). Data management for that Pilot Phase included collection, integration, analysis, and dissemination of findings from participating laboratories and data repositories. Many investigators face the same challenges of integration of data from complex, dynamic serum, and plasma specimens. The PPP workflow assembled a representative Core Dataset of 3,020 protein identifications, overcoming ambiguity and redundancy in the heterogeneous contributed identifications and redundancy and updates in the protein sequence databases. The results were made available with alternative thresholds from the University of Michigan, yielding a range of numbers of protein identifications. Data were submitted to EBI/PRIDE and to ISB/PeptideAtlas. The current phase of the PPP employs Proteome Xchange to link submission of well-annotated primary datasets to EBI/PRIDE, distributed file sharing by Tranche/ Proteome Commons.org, and reanalysis from the primary raw spectra at ISB/PeptideAtlas. Such human plasma proteome datasets are available for data mining comparisons with the proteomes of other organs and biofluids in health and disease. PMID:21063952

  5. Efficient quantitative comparisons of plasma proteomes using label-free analysis with MaxQuant

    PubMed Central

    Beer, Lynn A.; Liu, Pengyuan; Ky, Bonnie; Barnhart, Kurt T.; Speicher, David W.

    2017-01-01

    Mass spectrometry (MS)- based quantitation of plasma proteomes is challenging due to the extremely wide dynamic range and molecular heterogeneity of plasma samples. However, recent advances in technology, MS-instrumentation, and bioinformatics have enabled in-depth quantitative analyses of very complex proteomes, including plasma. Specifically, recent improvements in both label-based and label-free quantitation strategies have allowed highly accurate quantitative comparisons of expansive proteome datasets. Here we present a method for in-depth label-free analysis of human plasma samples using MaxQuant. PMID:28674895

  6. Characterization of human plasma proteome dynamics using deuterium oxide

    PubMed Central

    Wang, Ding; Liem, David A; Lau, Edward; Ng, Dominic CM; Bleakley, Brian J; Cadeiras, Martin; Deng, Mario C; Lam, Maggie PY; Ping, Peipei

    2016-01-01

    Purpose High-throughput quantification of human protein turnover via in vivo administration of deuterium oxide (2H2O) is a powerful new approach to examine potential disease mechanisms. Its immediate clinical translation is contingent upon characterizations of the safety and hemodynamic effects of in vivo administration of 2H2O to human subjects. Experimental design We recruited 10 healthy human subjects with a broad demographic variety to evaluate the safety, feasibility, efficacy, and reproducibility of 2H2O intake for studying protein dynamics. We designed a protocol where each subject orally consumed weight-adjusted doses of 70% 2H2O daily for 14 days to enrich body water and proteins with deuterium. Plasma proteome dynamics was measured using a high-resolution MS method we recently developed. Results This protocol was successfully applied in 10 human subjects to characterize the endogenous turnover rates of 542 human plasma proteins, the largest such human dataset to-date. Throughout the study, we did not detect physiological effects or signs of discomfort from 2H2O consumption. Conclusions and clinical relevance Our investigation supports the utility of a 2H2O intake protocol that is safe, accessible, and effective for clinical investigations of large-scale human protein turnover dynamics. This workflow shows promising clinical translational value for examining plasma protein dynamics in human diseases. PMID:24946186

  7. Establishing a leaf proteome reference map for Ginkgo biloba provides insight into potential ethnobotanical uses

    USDA-ARS?s Scientific Manuscript database

    Although ginkgo (Maidenhair tree, Ginkgo biloba L.) is an ancient medicinal and ornamental tree, there has not previously been any systematic proteomic study of the leaves. Herein we describe results from the initial study identifying abundant ginkgo leaf proteins and present a gel reference map. Pr...

  8. Large-scale multiplexed quantitative discovery proteomics enabled by the use of an (18)O-labeled "universal" reference sample.

    PubMed

    Qian, Wei-Jun; Liu, Tao; Petyuk, Vladislav A; Gritsenko, Marina A; Petritis, Brianne O; Polpitiya, Ashoka D; Kaushal, Amit; Xiao, Wenzhong; Finnerty, Celeste C; Jeschke, Marc G; Jaitly, Navdeep; Monroe, Matthew E; Moore, Ronald J; Moldawer, Lyle L; Davis, Ronald W; Tompkins, Ronald G; Herndon, David N; Camp, David G; Smith, Richard D

    2009-01-01

    The quantitative comparison of protein abundances across a large number of biological or patient samples represents an important proteomics challenge that needs to be addressed for proteomics discovery applications. Herein, we describe a strategy that incorporates a stable isotope (18)O-labeled "universal" reference sample as a comprehensive set of internal standards for analyzing large sample sets quantitatively. As a pooled sample, the (18)O-labeled "universal" reference sample is spiked into each individually processed unlabeled biological sample and the peptide/protein abundances are quantified based on (16)O/(18)O isotopic peptide pair abundance ratios that compare each unlabeled sample to the identical reference sample. This approach also allows for the direct application of label-free quantitation across the sample set simultaneously along with the labeling-approach (i.e., dual-quantitation) since each biological sample is unlabeled except for the labeled reference sample that is used as internal standards. The effectiveness of this approach for large-scale quantitative proteomics is demonstrated by its application to a set of 18 plasma samples from severe burn patients. When immunoaffinity depletion and cysteinyl-peptide enrichment-based fractionation with high resolution LC-MS measurements were combined, a total of 312 plasma proteins were confidently identified and quantified with a minimum of two unique peptides per protein. The isotope labeling data was directly compared with the label-free (16)O-MS intensity data extracted from the same data sets. The results showed that the (18)O reference-based labeling approach had significantly better quantitative precision compared to the label-free approach. The relative abundance differences determined by the two approaches also displayed strong correlation, illustrating the complementary nature of the two quantitative methods. The simplicity of including the (18)O-reference for accurate quantitation makes this

  9. Toward Plasma Proteome Profiling with Ion Mobility-Mass Spectrometry

    SciTech Connect

    Valentine, Stephen J.; Plasencia, Manolo D.; Liu, Xiaoyun; Krishnan, Meera; Naylor, Stephen; Udseth, Harold R.; Smith, Richard D.; Clemmer, David E.

    2006-11-01

    Differential, functional, and mapping proteomic analyses of complex biological mixtures suffer from a lack of component resolution. Here we describe the application of ion mobility-mass spectrometry (IMSMS) to this problem. With this approach, components that are separated by liquid chromatography are dispersed based on differences in their mobilities through a buffer gas prior to being analyzed by MS. The inclusion of the gas-phase dispersion provides more than an order of magnitude enhancement in component resolution at no cost to data acquisition time. Additionally, the mobility separation often removes high-abundance species from spectral regions containing low-abundance species, effectively increasing measurement sensitivity and dynamic range. Finally, collision-induced dissociation of all ions can be recorded in a single experimental sequence while conventional MS methods sequentially select precursors. The approach is demonstrated in a single, rapid (3.3 h) analysis of a plasma digest sample where abundant proteins have not been removed. Protein database searches have yielded 731 high confidence peptide assignments corresponding to 438 unique proteins. Results have been compiled into an initial analytical map to be used -after further augmentation and refinement- for comparative plasma profiling studies.

  10. Proteomic Changes in Chicken Plasma Induced by Salmonella typhimurium Lipopolysaccharides

    PubMed Central

    Packialakshmi, Balamurugan; Liyanage, Rohana; Lay, Jackson O.; Makkar, Sarbjeet K.; Rath, Narayan C.

    2016-01-01

    Lipopolysaccharides (LPS) are cell wall components of Gram-negative bacteria that produce inflammation and sickness in higher animals. The objective was to identify plasma proteomic changes in an avian model of inflammation. Chickens were treated with either saline or LPS, and blood was collected at 24 hours postinjection. The pooled plasma samples were depleted of high-abundant proteins and analyzed by matrix-assisted laser desorption ionization (MALDI)-time-of-flight mass spectrometry and liquid chromatography–tandem mass spectrometry (LC–MS/MS). MALDI analyses showed an increase in fibrinogen beta-derived peptide and a decrease in apolipoprotein-AII-derived peptide in LPS samples. Label-free quantitation of LC–MS/MS spectra revealed an increase in the levels of α1-acid glycoprotein, a chemokine CCLI10, and cathelicidin-2, but a decrease in an interferon-stimulated gene-12-2 protein in the LPS group. These differentially expressed proteins are associated with immunomodulation, cytokine changes, and defense mechanisms, which may be useful as candidate biomarkers of infection and inflammation. PMID:27053921

  11. Comparative proteome analysis of egg yolk plasma proteins during storage.

    PubMed

    Gao, Dan; Qiu, Ning; Liu, Yaping; Ma, Meihu

    2017-06-01

    Physical changes such as chicken egg white thinning and egg yolk flattening occur during storage, implying a decline in egg quality. To reveal the deteriorative process related to chicken egg internal quality, a comparative proteomic method was used in this study to analyze the alterations in egg yolk plasma proteins at different storage times (0, 20 and 40 days) under an ambient temperature of 22 ± 2 °C. Using two-dimensional electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry, 33 protein spots representing 12 proteins were identified with significant (P < 0.05) alterations in abundance at different storage times. The proteins that showed significant changes in abundance included serum albumin, vitellogenin fragments, IgY chains, ovalbumin, ovoinhibitor, α2 -macroglobulin-like protein 1-like, hemopexin, transthyretin, apolipoprotein A-I and β2 -glycoprotein I precursor. Accelerating degradation for most egg yolk plasma proteins was observed after prolonged storage (from day 20 to day 40). It is likely that the increased degradation of protease inhibitors such as ovoinhibitor and α2 -macroglobulin-like protein 1-like during prolonged storage lead to an imbalance of protease and antiprotease in egg yolk, which may play a key role in the degradation of egg yolk proteins. These findings will provide an insight into the effects of storage on egg yolk protein changes and give a deeper understanding of the deteriorative process of chicken egg yolk. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  12. Detailed tail proteomic analysis of axolotl (Ambystoma mexicanum) using an mRNA-seq reference database.

    PubMed

    Demircan, Turan; Keskin, Ilknur; Dumlu, Seda Nilgün; Aytürk, Nilüfer; Avşaroğlu, Mahmut Erhan; Akgün, Emel; Öztürk, Gürkan; Baykal, Ahmet Tarık

    2017-01-01

    Salamander axolotl has been emerging as an important model for stem cell research due to its powerful regenerative capacity. Several advantages, such as the high capability of advanced tissue, organ, and appendages regeneration, promote axolotl as an ideal model system to extend our current understanding on the mechanisms of regeneration. Acknowledging the common molecular pathways between amphibians and mammals, there is a great potential to translate the messages from axolotl research to mammalian studies. However, the utilization of axolotl is hindered due to the lack of reference databases of genomic, transcriptomic, and proteomic data. Here, we introduce the proteome analysis of the axolotl tail section searched against an mRNA-seq database. We translated axolotl mRNA sequences to protein sequences and annotated these to process the LC-MS/MS data and identified 1001 nonredundant proteins. Functional classification of identified proteins was performed by gene ontology searches. The presence of some of the identified proteins was validated by in situ antibody labeling. Furthermore, we have analyzed the proteome expressional changes postamputation at three time points to evaluate the underlying mechanisms of the regeneration process. Taken together, this work expands the proteomics data of axolotl to contribute to its establishment as a fully utilized model. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Emerging Affinity-Based Proteomic Technologies for Large-Scale Plasma Profiling in Cardiovascular Disease.

    PubMed

    Smith, J Gustav; Gerszten, Robert E

    2017-04-25

    Plasma biomarkers that reflect molecular states of the cardiovascular system are central for clinical decision making. Routinely used plasma biomarkers include troponins, natriuretic peptides, and lipoprotein particles, yet interrogate only a modest subset of pathways relevant to cardiovascular disease. Systematic profiling of a larger portion of circulating plasma proteins (the plasma proteome) will provide opportunities for unbiased discovery of novel markers to improve diagnostic or predictive accuracy. In addition, proteomic profiling may inform pathophysiological understanding and point to novel therapeutic targets. Obstacles for comprehensive proteomic profiling include the immense size and structural heterogeneity of the proteome, and the broad range of abundance levels, as well. Proteome-wide, untargeted profiling can be performed in tissues and cells with tandem mass spectrometry. However, applications to plasma are limited by the need for complex preanalytical sample preparation stages limiting sample throughput. Multiplexing of targeted methods based on capture and detection of specific proteins are therefore receiving increasing attention in plasma proteomics. Immunoaffinity assays are the workhorse for measuring individual proteins but have been limited for proteomic applications by long development times, cross-reactivity preventing multiplexing, specificity issues, and incomplete sensitivity to detect proteins in the lower range of the abundance spectrum (below picograms per milliliter). Emerging technologies to address these issues include nucleotide-labeled immunoassays and aptamer reagents that can be automated for efficient multiplexing of thousands of proteins at high sample throughput, coupling of affinity capture methods to mass spectrometry for improved specificity, and ultrasensitive detection systems to measure low-abundance proteins. In addition, proteomics can now be integrated with modern genomics tools to comprehensively relate

  14. Proteomic changes in plasma of broiler chickens with femoral head necrosis

    USDA-ARS?s Scientific Manuscript database

    Femoral head necrosis (FHN) is a skeletal problem in broiler chickens where the proximal femoral head cartilage shows susceptibility to separation from its growth plate. The FHN selected birds showed higher bodyweights and reduced plasma cholesterol. The proteomic differences in the plasma of health...

  15. Dietary zinc depletion and repletion affects plasma proteins: an analysis of the plasma proteome.

    PubMed

    Grider, Arthur; Wickwire, Kathie; Ho, Emily; Chung, Carolyn S; King, Janet

    2013-02-01

    Zinc (Zn) deficiency is a problem world-wide. Current methods for assessing Zn status are limited to measuring plasma or serum Zn within populations suspected of deficiency. Despite the high prevalence of Zn deficiency in the human population there are no methods currently available for sensitively assessing Zn status among individuals. The purpose of this research was to utilize a proteomic approach using two-dimensional gel electrophoresis (2DE) and mass spectrometry to identify protein biomarkers that were sensitive to changes in dietary Zn levels in humans. Proteomic analysis was performed in human plasma samples (n = 6) obtained from healthy adult male subjects that completed a dietary Zn depletion/repletion protocol, current dietary zinc intake has a greater effect on fractional zinc absorption than does longer term zinc consumption in healthy adult men. Chung et al. (Am J Clin Nutr 87 (5):1224-1229, 2008). After a 13 day Zn acclimatization period where subjects consumed a Zn-adequate diet, the male subjects consumed a marginal Zn-depleted diet for 42 days followed by consumption of a Zn-repleted diet for 28 days. The samples at baseline, end of depletion and end of repletion were pre-fractionated through immuno-affinity columns to remove 14 highly abundant proteins, and each fraction separated by 2DE. Following staining by colloidal Coomassie blue and densitometric analysis, three proteins were identified by mass spectrometry as affected by changes in dietary Zn. Fibrin β and chain E, fragment double D were observed in the plasma protein fraction that remained bound to the immunoaffinity column. An unnamed protein that was related to immunoglobulins was observed in the immunodepleted plasma fraction. Fibrin β increased two-fold following the Zn depletion period and decreased to baseline values following the Zn repletion period; this protein may serve as a viable biomarker for Zn status in the future.

  16. Reference map and comparative proteomic analysis of Neisseria gonorrhoeae displaying high resistance against spectinomycin.

    PubMed

    Nabu, Sunanta; Lawung, Ratana; Isarankura-Na-Ayudhya, Patcharee; Isarankura-Na-Ayudhya, Chartchalerm; Roytrakul, Sittiruk; Prachayasittikul, Virapong

    2014-03-01

    A proteome reference map of Neisseria gonorrhoeae was successfully established using two-dimensional gel electrophoresis in conjunction with matrix-assisted laser desorption ionization-time of flight mass spectrometry. This map was further applied to compare protein expression profiles of high-level spectinomycin-resistant (clinical isolate) and -susceptible (reference strain) N. gonorrhoeae following treatment with subminimal inhibitory concentrations (subMICs) of spectinomycin. Approximately 200 protein spots were visualized by Coomassie brilliant blue G-250 staining and 66 spots representing 58 unique proteins were subsequently identified. Most of the identified proteins were analysed as cytoplasmic proteins and belonged to the class of energy metabolism. Comparative proteomic analysis of whole protein expression of susceptible and resistant gonococci showed up to 96% similarity while eight proteins were found to be differentially expressed in the resistant strain. In the presence of subMICs of spectinomycin, it was found that 50S ribosomal protein L7/L12, an essential component for ribosomal translocation, was upregulated in both strains, ranging from 1.5- to 3.5-fold, suggesting compensatory mechanisms of N. gonorrhoeae in response to antibiotic that inhibits protein synthesis. Moreover, the differential expression of proteins involved in energy metabolism, amino acid biosynthesis, and the cell envelope was noticeably detected, indicating significant cellular responses and adaptation against antibiotic stress. Such knowledge provides valuable data, not only fundamental proteomic data, but also knowledge of the mode of action of antibiotic and secondary target proteins implicated in adaptation and compensatory mechanisms.

  17. Proteomic Investigation of Ram Spermatozoa and the Proteins Conferred by Seminal Plasma.

    PubMed

    Pini, Taylor; Leahy, Tamara; Soleilhavoup, Clement; Tsikis, Guillaume; Labas, Valerie; Combes-Soia, Lucie; Harichaux, Gregoire; Rickard, Jessica P; Druart, Xavier; de Graaf, Simon P

    2016-10-07

    Sperm proteomes have emerged for several species; however, the extent of species similarity is unknown. Sheep are an important agricultural species for which a comprehensive sperm proteome has not been produced. In addition, potential proteomic factors from seminal plasma that may contribute to improved fertility after cervical insemination are yet to be explored. Here we use liquid chromatography-tandem mass spectrometry to investigate the proteome of ejaculated ram spermatozoa, with quantitative comparison to epididymal spermatozoa. We also present a comparison to published proteomes of five other species. We identified 685 proteins in ejaculated ram spermatozoa, with the most abundant proteins involved in metabolic pathways. Only 5% of ram sperm proteins were not detected in other species, which suggest highly conserved structures and pathways. Of the proteins present in both epididymal and ejaculated ram spermatozoa, 7% were more abundant in ejaculated spermatozoa. Only two membrane-bound proteins were detected solely in ejaculated sperm lysates: liver enriched gene 1 (LEG1/C6orf58) and epidermal growth factor-like repeats and discoidin I-like domains 3 (EDIL3). This is the first evidence that despite its relatively complex proteomic composition, seminal plasma exposure leads to few novel proteins binding tightly to the ram sperm plasma membrane.

  18. 18O-Labeled Proteome Reference as Global Internal Standards for Targeted Quantification by Selected Reaction Monitoring-Mass Spectrometry

    SciTech Connect

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

    2011-10-11

    Selected reaction monitoring-mass spectrometry (SRM-MS) is an emerging technology for high throughput targeted protein quantification and verification in biological and biomarker discovery studies; however, the cost associated with the use of stable isotope labeled synthetic peptides as internal standards is prohibitive for quantitatively screening large numbers of candidate proteins as often required in the pre-verification phase of biomarker discovery. Herein we present the proof-of-concept experiments of using an 18O-labeled 'universal' reference as comprehensive internal standards for quantitative SRM-MS analysis. With an 18O-labeled whole proteome sample as reference, every peptide of interest will have its own corresponding heavy isotope labeled internal standard, thus providing an ideal approach for quantitative screening of a large number of candidates using SRM-MS. Our results showed that the 18O incorporation efficiency using a recently improved protocol was >99.5% for most peptides investigated, a level comparable to 13C/15N labeled synthetic peptides in terms of heavy isotope incorporation. The accuracy, reproducibility, and linear dynamic range of quantification were further assessed based on known ratios of standard proteins spiked into mouse plasma with an 18O-labeled mouse plasma reference. A dynamic range of four orders of magnitude in relative concentration was obtained with high reproducibility (i.e., coefficient of variance <10%) based on the 16O/18O peak area ratios. Absolute and relative quantification of C-reactive protein and prostate-specific antigen were demonstrated by coupling an 18O-labeled reference with standard additions of protein standards. Collectively, our results demonstrated that the use of 18O-labeled reference provides a convenient and effective strategy for quantitative SRM screening of large number of candidate proteins.

  19. Differential proteomics of human seminal plasma: A potential target for searching male infertility marker proteins.

    PubMed

    Tomar, Anil Kumar; Sooch, Balwinder Singh; Singh, Sarman; Yadav, Savita

    2012-04-01

    The clinical fertility tests, available in the market, fail to define the exact cause of male infertility in almost half of the cases and point toward a crucial need of developing better ways of infertility investigations. The protein biomarkers may help us toward better understanding of unknown cases of male infertility that, in turn, can guide us to find better therapeutic solutions. Many clinical attempts have been made to identify biomarkers of male infertility in sperm proteome but only few studies have targeted seminal plasma. Human seminal plasma is a rich source of proteins that are essentially required for development of sperm and successful fertilization. This viewpoint article highlights the importance of human seminal plasma proteome in reproductive physiology and suggests that differential proteomics integrated with functional analysis may help us in searching potential biomarkers of male infertility.

  20. Quantitative Trait Loci Mapping of the Mouse Plasma Proteome (pQTL)

    PubMed Central

    Holdt, Lesca M.; von Delft, Annette; Nicolaou, Alexandros; Baumann, Sven; Kostrzewa, Markus; Thiery, Joachim; Teupser, Daniel

    2013-01-01

    A current challenge in the era of genome-wide studies is to determine the responsible genes and mechanisms underlying newly identified loci. Screening of the plasma proteome by high-throughput mass spectrometry (MALDI-TOF MS) is considered a promising approach for identification of metabolic and disease processes. Therefore, plasma proteome screening might be particularly useful for identifying responsible genes when combined with analysis of variation in the genome. Here, we describe a proteomic quantitative trait locus (pQTL) study of plasma proteome screens in an F2 intercross of 455 mice mapped with 177 genetic markers across the genome. A total of 69 of 176 peptides revealed significant LOD scores (≥5.35) demonstrating strong genetic regulation of distinct components of the plasma proteome. Analyses were confirmed by mechanistic studies and MALDI-TOF/TOF, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of the two strongest pQTLs: A pQTL for mass-to-charge ratio (m/z) 3494 (LOD 24.9, D11Mit151) was identified as the N-terminal 35 amino acids of hemoglobin subunit A (Hba) and caused by genetic variation in Hba. Another pQTL for m/z 8713 (LOD 36.4; D1Mit111) was caused by variation in apolipoprotein A2 (Apoa2) and cosegregated with HDL cholesterol. Taken together, we show that genome-wide plasma proteome profiling in combination with genome-wide genetic screening aids in the identification of causal genetic variants affecting abundance of plasma proteins. PMID:23172855

  1. [Comparative proteome analysis of blood plasma of patients with early-stage chronic cerebral ischemia].

    PubMed

    Kisrieva, Y S; Petushkova, N A; Samenkova, N F; Kuznetsova, G P; Larina, O V; Zavialova, M G; Teryaeva, N B; Belyaev, A Y; Karuzina, I I

    2016-07-01

    In the present study, we explored the technology of liquid chromatography-mass spectrometry (HPLC-MS/MS) for the proteome analysis of blood plasma of patients with early chronic cerebral ischemia. Analysis of mass-spectrometer data carried out in automatic mode using the software Progenesis LS-MS. As a result of this study identified 43 proteins. The differences identified in the study group compared with the control in 7 proteins. It was found that in the early stages of chronic cerebral ischemia proteome changes in blood plasma affect proteins related to the immune system, the system for the maintenance of hemostasis and lipid metabolism.

  2. International Reference Ionosphere: Plasma densities - Status 1988

    NASA Technical Reports Server (NTRS)

    Rawer, K.; Bilitza, D.

    1990-01-01

    An account is given of the changes proposed in 1988 for the International Reference Ionosphere electron density profile, as well as the status of their implementation. The fully analytical profile function under development for the entire ionosphere can be achieved with a linear combination of several LAY functions. Although four LAY functions are required to describe the density features of the middle ionosphere, three LAY functions suffice to reproduce electron densities in both the topside ionosphere and lower ionosphere. Attention is given to the way in which the LAY parameters are computationally derivable from characteristic profile points.

  3. Tumor microenvironment-derived proteins dominate the plasma proteome response during breast cancer induction and progression.

    PubMed

    Pitteri, Sharon J; Kelly-Spratt, Karen S; Gurley, Kay E; Kennedy, Jacob; Buson, Tina Busald; Chin, Alice; Wang, Hong; Zhang, Qing; Wong, Chee-Hong; Chodosh, Lewis A; Nelson, Peter S; Hanash, Samir M; Kemp, Christopher J

    2011-08-01

    Tumor development relies upon essential contributions from the tumor microenvironment and host immune alterations. These contributions may inform the plasma proteome in a manner that could be exploited for cancer diagnosis and prognosis. In this study, we employed a systems biology approach to characterize the plasma proteome response in the inducible HER2/neu mouse model of breast cancer during tumor induction, progression, and regression. Mass spectrometry data derived from approximately 1.6 million spectra identified protein networks involved in wound healing, microenvironment, and metabolism that coordinately changed during tumor development. The observed alterations developed prior to cancer detection, increased progressively with tumor growth and reverted toward baseline with tumor regression. Gene expression and immunohistochemical analyses suggested that the cancer-associated plasma proteome was derived from transcriptional responses in the noncancerous host tissues as well as the developing tumor. The proteomic signature was distinct from a nonspecific response to inflammation. Overall, the developing tumor simultaneously engaged a number of innate physiologic processes, including wound repair, immune response, coagulation and complement cascades, tissue remodeling, and metabolic homeostasis that were all detectable in plasma. Our findings offer an integrated view of tumor development relevant to plasma-based strategies to detect and diagnose cancer.

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

    Cancer.gov

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

  5. Lymph is not a Plasma Ultrafiltrate: A Proteomic Analysis of Injured Patients

    PubMed Central

    Moore, Ernest E.; Wohlauer, Max; Banerjee, Anirban; Silliman, Christopher C; Hansen, Kirk C.

    2014-01-01

    Studies on animal models have documented a role for the water soluble protein fraction of mesenteric lymph as a conduit from hemorrhagic shock to acute lung injury and post-injury multiple organ failure. We hypothesize that mesenteric lymph is not an ultrafiltrate of plasma and contains specific protein mediators that may predispose patients to ALI/MOF. Mesenteric lymph and plasma were collected from critically ill or injured patients and from nine patients with lymphatic injuries, during semi-elective spine reconstruction, or immediately before organ donation. Proteomic analyses were performed through immuno-affinity depletion of the 14 most abundant plasma proteins, and GeLC-MS analyses. Overall, 548 proteins were identified in the patients undergoing semi-elective surgery, of which 155 were uniquely present in the lymph. In addition, the post-shock plasma proteome was characterized by peculiar features, suggesting that only a partial overlap exists between the plasma and mesenteric lymph from trauma patients. Differential proteins between the matched plasma and mesenteric lymph from trauma patients could be related to, coagulopathy and hypercoagulability, cell lysis, pro-inflammatory responses and immune system activation, extracellular matrix remodeling, lymph-specific immunomodulation and vascular hypoactivity/neoangiogenesis, and energy/redox metabolic adaptation to trauma. In conclusion, the proteome of mesenteric lymph is biologically different (in qualitative and quantitative terms) than that of a mere plasma ultrafiltrate. PMID:25243428

  6. A reference transcriptome and inferred proteome for the salamander Notophthalmus viridescens.

    PubMed

    Abdullayev, Ilgar; Kirkham, Matthew; Björklund, Åsa K; Simon, András; Sandberg, Rickard

    2013-05-01

    Salamanders have a remarkable capacity to regenerate complex tissues, such as limbs and brain, and are therefore an important comparative model system for regenerative medicine. Despite these unique properties among adult vertebrates, the genomic information for amphibians in general, and salamanders in particular, is scarce. Here, we used massive parallel sequencing to reconstruct a de novo reference transcriptome of the red spotted newt (Notophthalmus viridescens) containing 118,893 transcripts with a N50 length of 2016 nts. Comparisons to other vertebrates revealed a newt transcriptome that is comparable in size and characteristics to well-annotated vertebrate transcriptomes. Identification of putative open reading frames (ORFs) enabled us to infer a comprehensive proteome, including the annotation of 19,903 newt proteins. We used the identified domain architectures (DAs) to assign ORFs phylogenetic positions, which also revealed putative salamander specific proteins. The reference transcriptome and inferred proteome of the red spotted newt will facilitate the use of systematic genomic technologies for regeneration studies in salamanders and enable evolutionary analyses of vertebrate regeneration at the molecular level.

  7. Large-scale inference of protein tissue origin in gram-positive sepsis plasma using quantitative targeted proteomics

    PubMed Central

    Malmström, Erik; Kilsgård, Ola; Hauri, Simon; Smeds, Emanuel; Herwald, Heiko; Malmström, Lars; Malmström, Johan

    2016-01-01

    The plasma proteome is highly dynamic and variable, composed of proteins derived from surrounding tissues and cells. To investigate the complex processes that control the composition of the plasma proteome, we developed a mass spectrometry-based proteomics strategy to infer the origin of proteins detected in murine plasma. The strategy relies on the construction of a comprehensive protein tissue atlas from cells and highly vascularized organs using shotgun mass spectrometry. The protein tissue atlas was transformed to a spectral library for highly reproducible quantification of tissue-specific proteins directly in plasma using SWATH-like data-independent mass spectrometry analysis. We show that the method can determine drastic changes of tissue-specific protein profiles in blood plasma from mouse animal models with sepsis. The strategy can be extended to several other species advancing our understanding of the complex processes that contribute to the plasma proteome dynamics. PMID:26732734

  8. Large-scale inference of protein tissue origin in gram-positive sepsis plasma using quantitative targeted proteomics.

    PubMed

    Malmström, Erik; Kilsgård, Ola; Hauri, Simon; Smeds, Emanuel; Herwald, Heiko; Malmström, Lars; Malmström, Johan

    2016-01-06

    The plasma proteome is highly dynamic and variable, composed of proteins derived from surrounding tissues and cells. To investigate the complex processes that control the composition of the plasma proteome, we developed a mass spectrometry-based proteomics strategy to infer the origin of proteins detected in murine plasma. The strategy relies on the construction of a comprehensive protein tissue atlas from cells and highly vascularized organs using shotgun mass spectrometry. The protein tissue atlas was transformed to a spectral library for highly reproducible quantification of tissue-specific proteins directly in plasma using SWATH-like data-independent mass spectrometry analysis. We show that the method can determine drastic changes of tissue-specific protein profiles in blood plasma from mouse animal models with sepsis. The strategy can be extended to several other species advancing our understanding of the complex processes that contribute to the plasma proteome dynamics.

  9. Proteomic profiling of plasma in Huntington's disease reveals neuroinflammatory activation and biomarker candidates.

    PubMed

    Dalrymple, Annette; Wild, Edward J; Joubert, Richard; Sathasivam, Kirupa; Björkqvist, Maria; Petersén, Asa; Jackson, Graham S; Isaacs, Jeremy D; Kristiansen, Mark; Bates, Gillian P; Leavitt, Blair R; Keir, Geoff; Ward, Malcolm; Tabrizi, Sarah J

    2007-07-01

    Huntington's disease (HD) causes widespread CNS changes and systemic abnormalities including endocrine and immune dysfunction. HD biomarkers are needed to power clinical trials of potential treatments. We used multiplatform proteomic profiling to reveal plasma changes with HD progression. Proteins of interest were evaluated using immunoblotting and ELISA in plasma from 2 populations, CSF and R6/2 mice. The identified proteins demonstrate neuroinflammation in HD and warrant further investigation as possible biomarkers.

  10. Role of curcuminoids in ameliorating oxidative modification in β-thalassemia/Hb E plasma proteome.

    PubMed

    Weeraphan, Churat; Srisomsap, Chantragan; Chokchaichamnankit, Daranee; Subhasitanont, Pantipa; Hatairaktham, Suneerat; Charoensakdi, Ratiya; Panichkul, Narumol; Siritanaratkul, Noppadol; Fucharoen, Suthat; Svasti, Jisnuson; Kalpravidh, Ruchaneekorn W

    2013-03-01

    Thalassemic patients often exhibit high levels of oxidative stress and iron overload, which can lead to hazardous complications. Curcuminoids, extracted from the spice turmeric, are known to have antioxidant and iron-chelating properties and have been proposed as a potential upstream therapy of thalassemia. Here we have applied proteomic techniques to study the protein profile and oxidative damage in the plasma of β-thalassemia/Hb E patients before and after treatment with curcuminoids. In this study, 10 β-thalassemia/Hb E patients were treated with 500 mg curcuminoids daily for 12 months. The plasma protein profile and protein carbonyl content were determined at baseline, 6 and 12 months using two-dimensional fluorescence difference gel electrophoresis and carbonyl immunoblotting, respectively. Other hematological, clinical, and biochemical parameters were also analyzed. Twenty-six spots, identified as coagulation factors and proteins involved in iron homeostasis, showed significantly decreased intensity in thalassemic plasma, compared to those of normal subjects. Treatment with curcuminoids up-regulated the plasma levels of these proteins and reduced their oxidative damage. Serum non-transferrin bound iron, platelet factor-3 like activity, oxidative stress parameters and antioxidant enzymes were also improved after curcuminoids treatment. This study is the first proteomic study of plasma in the thalassemic state and also shows the ameliorating role of curcuminoids towards oxidative stress and iron overload in the plasma proteome.

  11. Plasma proteome analysis of patients with type 1 diabetes with diabetic nephropathy

    PubMed Central

    2010-01-01

    Background As part of a clinical proteomics program focused on diabetes and its complications we are looking for new and better protein biomarkers for diabetic nephropathy. The search for new and better biomarkers for diabetic nephropathy has, with a few exceptions, previously focused on either hypothesis-driven studies or urinary based investigations. To date only two studies have investigated the proteome of blood in search for new biomarkers, and these studies were conducted in sera from patients with type 2 diabetes. This is the first reported in depth proteomic study where plasma from type 1 diabetic patients was investigated with the goal of finding improved candidate biomarkers to predict diabetic nephropathy. In order to reach lower concentration proteins in plasma a pre-fractionation step, either hexapeptide bead-based libraries or anion exchange chromatography, was performed prior to surface enhanced laser desorption/ionization time-of-flight mass spectrometry analysis. Results Proteomic analysis of plasma from a cross-sectional cohort of 123 type 1 diabetic patients previously diagnosed as normoalbuminuric, microalbuminuric or macroalbuminuric, gave rise to 290 peaks clusters of which 16 were selected as the most promising biomarker candidates based on statistical performance, including independent component analysis. Four of the peaks that were discovered have been identified as transthyretin, apolipoprotein A1, apolipoprotein C1 and cystatin C. Several yet unidentified proteins discovered by this novel approach appear to have more potential as biomarkers for diabetic nephropathy. Conclusion These results demonstrate the capacity of proteomic analysis of plasma, by confirming the presence of known biomarkers as well as revealing new biomarkers for diabetic nephropathy in plasma in type 1 diabetic patients. PMID:20205888

  12. Proteomics-based identification of plasma biomarkers in oral squamous cell carcinoma.

    PubMed

    Tung, Chun-Liang; Lin, Szu-Ting; Chou, Hsiu-Chuan; Chen, Yi-Wen; Lin, Hwan-Chung; Tung, Chung-Liang; Huang, Kao-Jean; Chen, Yi-Ju; Lee, Ying-Ray; Chan, Hong-Lin

    2013-03-05

    Oral squamous cell carcinoma (OSCC) is an aggressive cancer and its occurrence is closely related to betel nut chewing in Taiwan. However, there are few prognostic and diagnostic biomarkers for this disease especially for its association with betel nut chewing. Recent progresses in quantitative proteomics have offered opportunities to discover plasma proteins as biomarkers for tracking the progression and for understanding the molecular mechanisms of OSCC. In present study, plasma samples from OSCC patients with at least 5-year history of betel nut chewing and healthy donors were analyzed by fluorescence 2D-DIGE-based proteomic analysis. Totally, 38 proteins have been firmly identified representing 13 unique gene products. These proteins mainly function in inflammatory responses (such as fibrinogen gamma chain) and transport (Apolipoprotein A-I). Additionally, the current quantitative proteomic approach has identified numerous OSCC biomarkers including fibrinogen (alpha/beta/gamma) chain, haptoglobin, leucine-rich alpha-2-glycoprotein and ribosomal protein S6 kinase alpha-3 (RSK2) which have not been reported and may be associated with the progression and development of the disease. In summary, this study reports a comprehensive patient-based proteomic approach for the identification of potential plasma biomarkers in OSCC. The potential of utilizing these markers for screening and treating OSCC warrants further investigations.

  13. Plasma proteome coverage is increased by unique peptide recovery from sodium deoxycholate precipitate.

    PubMed

    Serra, Aida; Zhu, Hongbin; Gallart-Palau, Xavier; Park, Jung Eun; Ho, Hee Haw; Tam, James P; Sze, Siu Kwan

    2016-03-01

    The ionic detergent sodium deoxycholate (SDC) is compatible with in-solution tryptic digestion and LC-MS/MS-based shotgun proteomics by virtue of being easy to separate from the peptide products via precipitation in acidic buffers. However, it remains unclear whether unique human peptides co-precipitate with SDC during acid treatment of complex biological samples. In this study, we demonstrate for the first time that a large quantity of unique peptides in human blood plasma can be co-precipitated with SDC using an optimized sample preparation method prior to shotgun proteomic analysis. We show that the plasma peptides co-precipitated with SDC can be successfully recovered using a sequential re-solubilization and precipitation procedure, and that this approach is particularly efficient at the extraction of long peptides. Recovery of peptides from the SDC pellet dramatically increased overall proteome coverage (>60 %), thereby improving the identification of low-abundance proteins and enhancing the identification of protein components of membrane-bound organelles. In addition, when we analyzed the physiochemical properties of the co-precipitated peptides, we observed that SDC-based sample preparation improved the identification of mildly hydrophilic/hydrophobic proteins that would otherwise be lost upon discarding the pellet. These data demonstrate that the optimized SDC protocol is superior to sodium dodecyl sulfate (SDS)/urea treatment for identifying plasma biomarkers by shotgun proteomics.

  14. Biological effects of static electric field: Plasma/serum proteome analysis of rats.

    PubMed

    Harutyunyan, Hayk; Artsruni, Gagik

    2013-03-01

    The external static electric field (SEF) of man-made origin brings to the substantially increased SEF background in a human environment the biological activity of which is a moot question. The paper reports on rats blood plasma/serum proteome modifications by means of 1D polyacrilamide gel electrophoresis and clotting process alterations after the short- and long-term SEF exposures of 200 kV/m. The results indicate decrease of fast α1 and α2 globular proteins in plasma coinciding with clotting acceleration after the short-term SEF, and attenuation of clotting-dependent proteome modifications reflected with incomplete coagulation after the long-term SEF exposure. Increased lysozyme activity in serum unlike plasma was observed after both SEF exposures. Applied model of the high-voltage SEF environment indicates dependence of biological systems functioning on the external SEF.

  15. An update on medium- and low-abundant blood plasma proteome of horse.

    PubMed

    Lepczyński, A; Ożgo, M; Dratwa-Chałupnik, A; Robak, P; Pyć, A; Zaborski, D; Herosimczyk, A

    2017-07-10

    The main objectives of the study were to: (1) deeply analyse the serum protein composition of Equus caballus, (2) assess the effectiveness of the high-abundant protein depletion and improve the concentration of medium- and low-abundant proteins. The analysis were performed on the blood plasma of three healthy part-Arabian mares. The implementation of two-dimensional electrophoresis and matrix-assisted laser desorption/ionisation - time of flight mass spectrometry allowed us to establish a horse plasma proteome map. Serum proteins were resolved at pH 4 to 7, followed by 12% SDS-PAGE. As a result 136 spots were successfully identified, representing the products of 46 unique genes. Of these, 22 gene products have not been previously identified in horse serum/plasma samples using proteomic tools. Gene ontology analysis showed that almost 30% of all identified gene products belong to the coagulation and complement cascades. These results can undoubtedly serve as a useful and prospective prerequisite for the future analysis of horse plasma proteome changes in different physiological and pathophysiological conditions. The use of the medium- and low-abundant protein enrichment tool increased their abundance and allowed us to identify a higher number of protein gene products. The highest depletion efficiency was observed for the most abundant plasma proteins, that is albumin, IgG heavy chains and serotransferrin.

  16. 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

  17. Rapid detection of proteins in transgenic crops without protein reference standards by targeted proteomic mass spectrometry.

    PubMed

    Schacherer, Lindsey J; Xie, Weiping; Owens, Michaela A; Alarcon, Clara; Hu, Tiger X

    2016-09-01

    Liquid chromatography coupled with tandem mass spectrometry is increasingly used for protein detection for transgenic crops research. Currently this is achieved with protein reference standards which may take a significant time or efforts to obtain and there is a need for rapid protein detection without protein reference standards. A sensitive and specific method was developed to detect target proteins in transgenic maize leaf crude extract at concentrations as low as ∼30 ng mg(-1) dry leaf without the need of reference standards or any sample enrichment. A hybrid Q-TRAP mass spectrometer was used to monitor all potential tryptic peptides of the target proteins in both transgenic and non-transgenic samples. The multiple reaction monitoring-initiated detection and sequencing (MIDAS) approach was used for initial peptide/protein identification via Mascot database search. Further confirmation was achieved by direct comparison between transgenic and non-transgenic samples. Definitive confirmation was provided by running the same experiments of synthetic peptides or protein standards, if available. A targeted proteomic mass spectrometry method using MIDAS approach is an ideal methodology for detection of new proteins in early stages of transgenic crop research and development when neither protein reference standards nor antibodies are available. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  18. 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

  19. The starch granule associated proteomes of commercially purified starch reference materials from rice and maize.

    PubMed

    Koziol, Adam G; Marquez, Benazir K; Huebsch, Matthew P; Smith, Jeffrey C; Altosaar, Illimar

    2012-01-04

    Commercially available reference materials are integral components of many experimental protocols, as it is critical to compare one's results to those derived from well-characterized standards. Most reference materials are well defined, with all their components being cataloged. However, certain reference materials, such as commercially prepared starch samples, can have undefined components, potentially limiting their usefulness as standards. The proteome of commercially prepared starch has not been documented, and to that end, we initiated a mass spectrometry-based survey of the proteins associated with starch granules in commercially prepared rice and maize starch samples. We performed direct trypsin treatments of starch samples and sequenced both the water-soluble peptides liberated into the aqueous supernatant and the peptides released from the starch granule surface by isopropanol solvent washing. We discovered that the majority of proteins, in both rice and maize samples, were involved in either carbohydrate metabolism or storage. We also documented proteins that are markers for seed maturity and for starch mobilization. Copyright © 2011 Elsevier B.V. All rights reserved.

  20. Plasma proteomic alterations in non-human primates and humans after chronic alcohol self-administration

    PubMed Central

    Freeman, Willard M.; VanGuilder, Heather D.; Guidone, Elizabeth; Krystal, John H.; Grant, Kathleen A.; Vrana, Kent E.

    2011-01-01

    Objective diagnostics of excessive alcohol use are valuable tools in the identification and monitoring of subjects with alcohol use disorders. A number of potential biomarkers of alcohol intake have been proposed, but none have reached widespread clinical usage, often due to limited diagnostic sensitivity and specificity. In order to identify novel potential biomarkers, we performed proteomic biomarker target discovery in plasma samples from non-human primates that chronically self-administer high levels of ethanol. 2-dimensional in-gel electrophoresis (2D-DIGE) was used to quantify plasma proteins from within subject samples collected before exposure to ethanol and after three months of excessive ethanol self-administration. Highly abundant plasma proteins were depleted from plasma samples to increase proteomic coverage. Altered plasma levels of SAA4, RBP, ITIH4, clusterin, and fibronectin, identified by 2D-DIGE analysis, were confirmed in unmanipulated, whole plasma from these animals by immunoblotting. Examination of these target plasma proteins in human subjects with excessive alcohol consumption (and control subjects) revealed increased levels of SAA4 and clusterin and decreased levels of fibronectin compared to controls. These proteins not only serve as targets for further development as biomarker candidates or components of biomarker panels, but also add to the growing understanding of dysregulated immune function and lipoprotein metabolism with chronic, excessive alcohol consumption. PMID:21303580

  1. A proteomic approach for plasma biomarker discovery with iTRAQ labelling and OFFGEL fractionation.

    PubMed

    Ernoult, Emilie; Bourreau, Anthony; Gamelin, Erick; Guette, Catherine

    2010-01-01

    Human blood plasma contains a plethora of proteins, encompassing not only proteins that have plasma-based functionalities, but also possibly every other form of low concentrated human proteins. As it circulates through the tissues, the plasma picks up proteins that are released from their origin due to physiological events such as tissue remodeling and cell death. Specific disease processes or tumors are often characterized by plasma "signatures," which may become obvious via changes in the plasma proteome profile, for example, through over expression of proteins. However, the wide dynamic range of proteins present in plasma makes their analysis very challenging, because high-abundance proteins tend to mask those of lower abundance. In the present study, we used a strategy combining iTRAQ as a reagent which improved the peptide ionization and peptide OFFGEL fractionation that has already been shown, in our previous research, to improve the proteome coverage of cellular extracts. Two prefractioning methods were compared: immunodepletion and a bead-based library of combinatorial hexapeptide technology. Our data suggested that both methods were complementary, with regard to the number of identified proteins. iTRAQ labelling, in association with OFFGEL fractionation, allowed more than 300 different proteins to be characterized from 400 microg of plasma proteins.

  2. Plasma proteomic alterations in non-human primates and humans after chronic alcohol self-administration.

    PubMed

    Freeman, Willard M; Vanguilder, Heather D; Guidone, Elizabeth; Krystal, John H; Grant, Kathleen A; Vrana, Kent E

    2011-08-01

    Objective diagnostics of excessive alcohol use are valuable tools in the identification and monitoring of subjects with alcohol use disorders. A number of potential biomarkers of alcohol intake have been proposed, but none have reached widespread clinical usage, often due to limited diagnostic sensitivity and specificity. In order to identify novel potential biomarkers, we performed proteomic biomarker target discovery in plasma samples from non-human primates that chronically self-administer high levels of ethanol. Two-dimensional difference in-gel electrophoresis (2D-DIGE) was used to quantify plasma proteins from within-subject samples collected before exposure to ethanol and after 3 months of excessive ethanol self-administration. Highly abundant plasma proteins were depleted from plasma samples to increase proteomic coverage. Altered plasma levels of serum amyloid A4 (SAA4), retinol-binding protein, inter-alpha inhibitor H4, clusterin, and fibronectin, identified by 2D-DIGE analysis, were confirmed in unmanipulated, whole plasma from these animals by immunoblotting. Examination of these target plasma proteins in human subjects with excessive alcohol consumption (and control subjects) revealed increased levels of SAA4 and clusterin and decreased levels of fibronectin compared to controls. These proteins not only serve as targets for further development as biomarker candidates or components of biomarker panels, but also add to the growing understanding of dysregulated immune function and lipoprotein metabolism with chronic, excessive alcohol consumption.

  3. A multiplex targeted proteomic assay for biomarker detection in plasma: a pancreatic cancer biomarker case study

    PubMed Central

    Pan, Sheng; Chen, Ru; Brand, Randall E; Hawley, Sarah; Tamura, Yasuko; Gafken, Philip R.; Milless, Brian P.; Goodlett, David R.; Rush, John; Brentnall, Teresa A.

    2012-01-01

    Biomarkers are most frequently proteins that are measured in the blood. Their development largely relies on antibody creation to test the protein candidate performance in blood samples of diseased versus non-diseased patients. The creation of such antibody assays has been a bottleneck in biomarker progress due to the cost, extensive time and effort required to complete the task. Targeted proteomics is an emerging technology that is playing an increasingly important role to facilitate disease biomarker development. In this study, we applied a SRM-based targeted proteomics platform to directly detect candidate biomarker proteins in plasma to evaluate their clinical utility for pancreatic cancer detection. The characterization of these protein candidates used a clinically well-characterized cohort that included plasma samples from patients with pancreatic cancer, chronic pancreatitis and healthy age-matched controls. Three of the five candidate proteins, including gelsolin, lumican and tissue inhibitor of metalloproteinase 1, demonstrated an AUC value greater than 0.75 in distinguishing pancreatic cancer from the controls. In addition, we provide an analysis of the reproducibility, accuracy, and robustness of the SRM-based proteomics platform. This information addresses important technical issues that could aid in the adoption of the targeted proteomics platform for practical clinical utility. PMID:22316387

  4. The Human Plasma Proteome Draft of 2017: Building on the Human Plasma PeptideAtlas from Mass Spectrometry and Complementary Assays.

    PubMed

    Schwenk, Jochen M; Omenn, Gilbert S; Sun, Zhi; Campbell, David S; Baker, Mark S; Overall, Christopher M; Aebersold, Ruedi; Moritz, Robert L; Deutsch, Eric W

    2017-09-22

    Human blood plasma provides a highly accessible window to the proteome of any individual in health and disease. Since its inception in 2002, the Human Proteome Organization's Human Plasma Proteome Project (HPPP) has been promoting advances in the study and understanding of the full protein complement of human plasma and on determining the abundance and modifications of its components. In 2017, we review the history of the HPPP and the advances of human plasma proteomics in general, including several recent achievements. We then present the latest 2017-04 build of Human Plasma PeptideAtlas, which yields ~43 million peptide-spectrum matches and 122,730 distinct peptide sequences from 178 individual experiments at a 1% protein-level FDR globally across all experiments. Applying the latest Human Proteome Project Data Interpretation Guidelines, we catalog 3509 proteins that have at least two non-nested uniquely-mapping peptides of 9 amino acids or more and >1300 additional proteins with ambiguous evidence. We apply the same two-peptide guideline to historical PeptideAtlas builds going back to 2006 and examine the progress made in the past ten years in plasma proteome coverage. We also compare the distribution of proteins in historical PeptideAtlas builds in various RNA-abundance and cellular localization categories. We then discuss advances in plasma proteomics based on targeted mass spectrometry as well as affinity assays, which during early 2017 target ~2000 proteins. Finally we describe considerations about sample handling and study design, concluding with an outlook for future advances in deciphering the human plasma proteome.

  5. Exploring the Human Seminal Plasma Proteome: An Unexplored Gold Mine of Biomarker for Male Infertility and Male Reproduction Disorder

    PubMed Central

    Gilany, Kambiz; Minai-Tehrani, Arash; Savadi-Shiraz, Elham; Rezadoost, Hassan; Lakpour, Niknam

    2015-01-01

    Background The human seminal fluid is a complex body fluid. It is not known how many proteins are expressed in the seminal plasma; however in analog with the blood it is possible up to 10,000 proteins are expressed in the seminal plasma. The human seminal fluid is a rich source of potential biomarkers for male infertility and reproduction disorder. Methods In this review, the ongoing list of proteins identified from the human seminal fluid was collected. To date, 4188 redundant proteins of the seminal fluid are identified using different proteomics technology, including 2-DE, SDS-PAGE-LC-MS/MS, MudPIT. However, this was reduced to a database of 2168 non-redundant protein using UniProtKB/Swiss-Prot reviewed database. Results The core concept of proteome were analyzed including pI, MW, Amino Acids, Chromosome and PTM distribution in the human seminal plasma proteome. Additionally, the biological process, molecular function and KEGG pathway were investigated using DAVID software. Finally, the biomarker identified in different male reproductive system disorder was investigated using proteomics platforms so far. Conclusion In this study, an attempt was made to update the human seminal plasma proteome database. Our finding showed that human seminal plasma studies used to date seem to have converged on a set of proteins that are repeatedly identified in many studies and that represent only a small fraction of the entire human seminal plasma proteome. PMID:25927022

  6. 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

  7. Plasma Proteome Association and Catalytic Activity of Stealth Polymer-Grafted Iron Oxide Nanoparticles.

    PubMed

    Wang, Miaoyi; Siddiqui, Ghizal; Gustafsson, Ove J R; Käkinen, Aleksandr; Javed, Ibrahim; Voelcker, Nicolas H; Creek, Darren J; Ke, Pu Chun; Davis, Thomas P

    2017-09-01

    Polyethylene glycol (PEG) is widely used as an antifouling and stealth polymer in surface engineering and nanomedicine. However, recent research has revealed adverse effects of bioaccumulation and immunogenicity following the administration of PEG, prompting this proteomic examination of the plasma protein coronae association with superparamagnetic iron oxide nanoparticles (IONPs) grafted with brushed PEG (bPEG) and an alternative, brushed phosphorylcholine (bPC). Using label-free quantitation by liquid chromatography tandem-mass spectrometry, this study determines protein abundances for the in vitro hard coronae of bare, bPC-, and bPEG-grafted IONPs in human plasma. This study also shows unique protein compositions in the plasma coronae of each IONP, including enrichment of coagulation factors and immunogenic complement proteins with bPEG, and enhanced binding of apolipoproteins with bPC. Functional analysis reveals that plasma protein coronae elevate the horseradish peroxidase-like activities of the bPC- and bPEG-IONPs by approximately twofold, an effect likely mediated by the diverse composition and physicochemical properties of the polymers as well as their associated plasma proteins. Taken together, these observations support the rational design of stealth polymers based on a quantitative understanding of the interplay between IONPs and the plasma proteome, and should prove beneficial for the development of materials for nanomedicine, biosensing, and catalysis. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Computational biomarker pipeline from discovery to clinical implementation: plasma proteomic biomarkers for cardiac transplantation.

    PubMed

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

    2013-04-01

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

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

    PubMed Central

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

    2013-01-01

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

  10. Affinity depletion of plasma and serum for mass spectrometry-based proteome analysis.

    PubMed

    Jaros, Julian A J; Guest, Paul C; Bahn, Sabine; Martins-de-Souza, Daniel

    2013-01-01

    Protein biomarker discovery in blood plasma and serum is severely hampered by the vast dynamic range of the proteome. With protein concentrations spanning 12 orders of magnitude, conventional mass spectrometric analysis allows for detection of only a few low-abundance proteins. Prior depletion of high-abundant proteins from the sample can increase analytical depth considerably and has become a widely used practice. We describe in detail an affinity depletion method that selectively removes 14 of the most abundant proteins in plasma and serum.

  11. Reference proteome of highly purified human Th1 cells reveals strong effects on metabolism and protein ubiquitination upon differentiation.

    PubMed

    Pagani, Massimiliano; Rockstroh, Maxie; Schuster, Maj; Rossetti, Grazisa; Moro, Monica; Crosti, Mariacristina; Tomm, Janina M

    2015-11-01

    The differentiation of human CD4(+) T cells into T helper cell subtypes and regulatory T cells is crucial to the immune response. Among subtypes, Th1 cells are dominant, representing approximately 50% of all lymphocytes. Thus far, most global proteomic studies have used only partially purified T helper cell subpopulations and/or have employed artificial protocols for inducing specific T helper cell subtypes and/or used gel-based approaches. These studies have shed light on molecular details of certain aspects of the proteome; nevertheless a global analysis of high purity primary naïve and Th1 cells by LC-MS/MS is required to provide a reference dataset for proteome-based T cell subtype characterization. The utilization of highly purified Th1 cells for a global proteome assessment and the bioinformatic comparison to naïve cells reveals changes in cell metabolism and the ubiquitination pathway upon T cell differentiation. All MS data have been deposited in the ProteomeXchange with identifier PXD001066 (http://proteomecentral.proteomexchange.org/dataset/PXD001066). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Exploring the Human Plasma Proteome for Humoral Mediators of Remote Ischemic Preconditioning - A Word of Caution

    PubMed Central

    Helgeland, Erik; Breivik, Lars Ertesvåg; Vaudel, Marc; Svendsen, Øyvind Sverre; Garberg, Hilde; Nordrehaug, Jan Erik; Berven, Frode Steingrimsen; Jonassen, Anne Kristine

    2014-01-01

    Despite major advances in early revascularization techniques, cardiovascular diseases are still the leading cause of death worldwide, and myocardial infarctions contribute heavily to this. Over the past decades, it has become apparent that reperfusion of blood to a previously ischemic area of the heart causes damage in and of itself, and that this ischemia reperfusion induced injury can be reduced by up to 50% by mechanical manipulation of the blood flow to the heart. The recent discovery of remote ischemic preconditioning (RIPC) provides a non-invasive approach of inducing this cardioprotection at a distance. Finding its endogenous mediators and their operative mode is an important step toward increasing the ischemic tolerance. The release of humoral factor(s) upon RIPC was recently demonstrated and several candidate proteins were published as possible mediators of the cardioprotection. Before clinical applicability, these potential biomarkers and their efficiency must be validated, a task made challenging by the large heterogeneity in reported data and results. Here, in an attempt to reproduce and provide more experimental data on these mediators, we conducted an unbiased in-depth analysis of the human plasma proteome before and after RIPC. From the 68 protein markers reported in the literature, only 28 could be mapped to manually reviewed (Swiss-Prot) protein sequences. 23 of them were monitored in our untargeted experiment. However, their significant regulation could not be reproducibly estimated. In fact, among the 394 plasma proteins we accurately quantified, no significant regulation could be confidently and reproducibly assessed. This indicates that it is difficult to both monitor and reproduce published data from experiments exploring for RIPC induced plasma proteomic regulations, and suggests that further work should be directed towards small humoral factors. To simplify this task, we made our proteomic dataset available via ProteomeXchange, where

  13. Proteomic profiling of human plasma exosomes identifies PPARgamma as an exosome-associated protein.

    PubMed

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

    2009-01-16

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

  14. Proteomic-Biostatistic Integrated Approach for Finding the Underlying Molecular Determinants of Hypertension in Human Plasma.

    PubMed

    Gajjala, Prathibha R; Jankowski, Vera; Heinze, Georg; Bilo, Grzegorz; Zanchetti, Alberto; Noels, Heidi; Liehn, Elisa; Perco, Paul; Schulz, Anna; Delles, Christian; Kork, Felix; Biessen, Erik; Narkiewicz, Krzysztof; Kawecka-Jaszcz, Kalina; Floege, Juergen; Soranna, Davide; Zidek, Walter; Jankowski, Joachim

    2017-08-01

    Despite advancements in lowering blood pressure, the best approach to lower it remains controversial because of the lack of information on the molecular basis of hypertension. We, therefore, performed plasma proteomics of plasma from patients with hypertension to identify molecular determinants detectable in these subjects but not in controls and vice versa. Plasma samples from hypertensive subjects (cases; n=118) and controls (n=85) from the InGenious HyperCare cohort were used for this study and performed mass spectrometric analysis. Using biostatistical methods, plasma peptides specific for hypertension were identified, and a model was developed using least absolute shrinkage and selection operator logistic regression. The underlying peptides were identified and sequenced off-line using matrix-assisted laser desorption ionization orbitrap mass spectrometry. By comparison of the molecular composition of the plasma samples, 27 molecular determinants were identified differently expressed in cases from controls. Seventy percent of the molecular determinants selected were found to occur less likely in hypertensive patients. In cross-validation, the overall R(2) was 0.434, and the area under the curve was 0.891 with 95% confidence interval 0.8482 to 0.9349, P<0.0001. The mean values of the cross-validated proteomic score of normotensive and hypertensive patients were found to be -2.007±0.3568 and 3.383±0.2643, respectively, P<0.0001. The molecular determinants were successfully identified, and the proteomic model developed shows an excellent discriminatory ability between hypertensives and normotensives. The identified molecular determinants may be the starting point for further studies to clarify the molecular causes of hypertension. © 2017 American Heart Association, Inc.

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

    PubMed Central

    Lee, Sun Eun; West, Keith P.; Cole, Robert N.; Schulze, Kerry J.; Christian, Parul; Wu, Lee Shu-Fune; Yager, James D.; Groopman, John; Ruczinski, Ingo

    2015-01-01

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

  16. Evaluating the effects of preanalytical variables on the stability of the human plasma proteome

    PubMed Central

    Hassis, Maria E.; Niles, Richard K.; Braten, Miles N.; Albertolle, Matthew E.; Witkowska, H. Ewa; Hubel, Carl A.; Fisher, Susan J.; Williams, Katherine E.

    2015-01-01

    High quality clinical biospecimens are vital for biomarker discovery, verification, and validation. Variations in blood processing and handling can affect protein abundances and assay reliability. Using an untargeted LC-MS approach, we systematically measured the impact of preanalytical variables on the plasma proteome. Time prior to processing was the only variable that affected the plasma protein levels. LC-MS quantification showed that preprocessing times <6 h had minimal effects on the immunodepleted plasma proteome, but by 4 days significant changes were apparent. Elevated levels of many proteins were observed, suggesting that in addition to proteolytic degradation during the preanalytical phase, changes in protein structure are also important considerations for protocols using antibody depletion. As to processing variables, a comparison of single- vs double-spun plasma showed minimal differences. After processing, the impact ≤3 freeze–thaw cycles was negligible regardless of whether freshly collected samples were processed in short succession or the cycles occurred during 14–17 years of frozen storage (−80 °C). Thus, clinical workflows that necessitate modest delays in blood processing times or employ different centrifugation steps can yield valuable samples for biomarker discovery and verification studies. PMID:25769420

  17. Low-Molecular-Weight Plasma Proteome Analysis Using Top-Down Mass Spectrometry.

    PubMed

    Cheon, Dong Huey; Yang, Eun Gyeong; Lee, Cheolju; Lee, Ji Eun

    2017-01-01

    While human plasma has a wealth of diagnostic information regarding the state of the human body in heath and disease, low molecular weight (LMW) proteome (<30 kDa) has been shown to contain a rich source of diagnostic biomarkers. Here we describe a protocol for top-down proteomic analysis to identify and characterize the LMW proteoforms present in four types of human plasma samples without immunoaffinity depletion and with depletion of the top two, six, and seven high-abundance proteins. Each type of plasma sample was first fractionated based on molecular weight using gel-eluted liquid fraction entrapment electrophoresis (GELFrEE). Then, the GELFrEE fractions containing up to 30 kDa were subjected to nanocapillary-LC-MS/MS, and the high-resolution MS and MS/MS data were processed using ProSightPC software. As a result, a total of 442 LMW proteins and cleaved products, including those with posttranslational modifications (PTMs) and single amino acid variations (SAAVs), were identified with a threshold E-value of 1 × 10(-4) from the four types of plasma samples.

  18. An Insight into the Changes in Human Plasma Proteome on Adaptation to Hypobaric Hypoxia

    PubMed Central

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

    2013-01-01

    Adaptation to hypobaric hypoxia is required by animals and human in several physiological and pathological situations. Hypobaric hypoxia is a pathophysiological condition triggering redox status disturbances of cell organization leading, via oxidative stress, to proteins, lipids, and DNA damage. Identifying the molecular variables playing key roles in this process would be of paramount importance to shed light on the mechanisms known to counteract the negative effects of oxygen lack. To obtain a molecular signature, changes in the plasma proteome were studied by using proteomic approach. To enrich the low-abundance proteins in human plasma, two highly abundant proteins, albumin and IgG, were first removed. By comparing the plasma proteins of high altitude natives with those of a normal control group, several proteins with a significant alteration were found. The up-regulated proteins were identified as vitamin D-binding protein, hemopexin, alpha-1–antitrypsin, haptoglobin β-chain, apolipoprotein A1, transthyretin and hemoglobin beta chain. The down-regulated proteins were transferrin, complement C3, serum amyloid, complement component 4A and plasma retinol binding protein. Among these proteins, the alterations of transthyretin and transferrin were further confirmed by ELISA and Western blotting analysis. Since all the up- and down- regulated proteins identified above are well-known inflammation inhibitors and play a positive anti-inflammatory role, these results show that there is some adaptive mechanism that sustains the inflammation balance in high altitude natives exposed to hypobaric hypoxia. PMID:23844025

  19. Rapid Response of the Yeast Plasma Membrane Proteome to Salt Stress*

    PubMed Central

    Szopinska, Aleksandra; Degand, Hervé; Hochstenbach, Jean-François; Nader, Joseph; Morsomme, Pierre

    2011-01-01

    The plasma membrane separates the cell from the external environment and plays an important role in the stress response of the cell. In this study, we compared plasma membrane proteome modifications of yeast cells exposed to mild (0.4 m NaCl) or high (1 m NaCl) salt stress for 10, 30, or 90 min. Plasma membrane-enriched fractions were isolated, purified, and subjected to iTRAQ labeling for quantitative analysis. In total, 88–109 plasma membrane proteins were identified and quantified. The quantitative analysis revealed significant changes in the abundance of several plasma membrane proteins. Mild salt stress caused an increase in abundance of 12 plasma membrane proteins, including known salt-responsive proteins, as well as new targets. Interestingly, 20 plasma membrane proteins, including the P-type H+-ATPase Pma1, ABC transporters, glucose and amino acid transporters, t-SNAREs, and proteins involved in cell wall biogenesis showed a significant and rapid decrease in abundance in response to both 0.4 m and 1 m NaCl. We propose that rapid protein internalization occurs as a response to hyper-osmotic and/or ionic shock, which might affect plasma membrane morphology and ionic homeostasis. This rapid response might help the cell to survive until the transcriptional response takes place. PMID:21825281

  20. RNAi-mediated downregulation of poplar plasma membrane intrinsic proteins (PIPs) changes plasma membrane proteome composition and affects leaf physiology.

    PubMed

    Bi, Zhen; Merl-Pham, Juliane; Uehlein, Norbert; Zimmer, Ina; Mühlhans, Stefanie; Aichler, Michaela; Walch, Axel Karl; Kaldenhoff, Ralf; Palme, Klaus; Schnitzler, Jörg-Peter; Block, Katja

    2015-10-14

    Plasma membrane intrinsic proteins (PIPs) are one subfamily of aquaporins that mediate the transmembrane transport of water. To reveal their function in poplar, we generated transgenic poplar plants in which the translation of PIP genes was downregulated by RNA interference investigated these plants with a comprehensive leaf plasma membrane proteome and physiome analysis. First, inhibition of PIP synthesis strongly altered the leaf plasma membrane protein composition. Strikingly, several signaling components and transporters involved in the regulation of stomatal movement were differentially regulated in transgenic poplars. Furthermore, hormonal crosstalk related to abscisic acid, auxin and brassinosteroids was altered, in addition to cell wall biosynthesis/cutinization, the organization of cellular structures and membrane trafficking. A physiological analysis confirmed the proteomic results. The leaves had wider opened stomata and higher net CO2 assimilation and transpiration rates as well as greater mesophyll conductance for CO2 (gm) and leaf hydraulic conductance (Kleaf). Based on these results, we conclude that PIP proteins not only play essential roles in whole leaf water and CO2 flux but have important roles in the regulation of stomatal movement. Copyright © 2015. Published by Elsevier B.V.

  1. Proteome reference map of Lactobacillus acidophilus NCFM and quantitative proteomics towards understanding the prebiotic action of lactitol.

    PubMed

    Majumder, Avishek; Sultan, Abida; Jersie-Christensen, Rosa R; Ejby, Morten; Schmidt, Bjarne Gregers; Lahtinen, Sampo J; Jacobsen, Susanne; Svensson, Birte

    2011-09-01

    Lactobacillus acidophilus NCFM is a probiotic bacterium adapted to survive in the gastrointestinal tract and with potential health benefits to the host. Lactitol is a synthetic sugar alcohol used as a sugar replacement in low calorie foods and selectively stimulating growth of L. acidophilus NCFM. In the present study the whole-cell extract proteome of L. acidophilus NCFM grown on glucose until late exponential phase was resolved by 2-DE (pH 3-7). A total of 275 unique proteins assigned to various physiological processes were identified from 650 spots. Differential 2-DE (DIGE) (pH 4-7) of L. acidophilus NCFM grown on glucose and lactitol, revealed 68 spots with modified relative intensity. Thirty-two unique proteins were identified in 41 of these spots changing 1.6-12.7-fold in relative abundance by adaptation of L. acidophilus NCFM to growth on lactitol. These proteins included β-galactosidase small subunit, galactokinase, galactose-1-phosphate uridylyltransferase and UDP-glucose-4-epimerase, which all are potentially involved in lactitol metabolism. This first comprehensive proteome analysis of L. acidophilus NCFM provides insights into protein abundance changes elicited by the prebiotic lactitol. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Plasma Proteomics Biomarkers in Alzheimer's Disease: Latest Advances and Challenges.

    PubMed

    Perneczky, Robert; Guo, Liang-Hao

    2016-01-01

    The recent paradigm shift towards a more biologically oriented definition of Alzheimer's disease (AD) in clinical settings increases the need for sensitive biomarkers that can be applied in population-based settings. Blood plasma is easily accessible and contains a large number of proteins related to cerebral processes. It is therefore an ideal candidate for AD biomarker discovery. The present chapter provides an overview of the current research landscape in relation to blood-based AD biomarkers. Both clinical and methodological issues are covered. A brief summary is given on two relevant laboratory techniques to ascertain blood biomarker changes due to AD; methodological and clinical challenges in the field are also discussed.

  3. Hemoglobin Depletion from plasma: Considerations for Proteomic Discovery in Sickle Cell Disease and other Hemolytic Processes

    PubMed Central

    Williams, Lisa M.; Fu, Zongming; Dulloor, Pratima; Yen, Timothy; Barron-Casella, Emily; Savage, William; Van Eyk, Jennifer E.; Casella, James F.; Everett, Allen

    2015-01-01

    Purpose Hemoglobin (Hb) depletion with nickel affinity chromatography has been shown to increase the number of proteins identified in proteomic studies of erythrocytes, but limited data exist on the application of this technique in depletion of Hb from plasma or serum required for clinical biomarker studies. The aim of this study was to explore the potential of using nickel-beads for Hb depletion of plasma. Experimental design Nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography was used to deplete Hb from hemolyzed plasma samples obtained from children with sickle cell disease (SCD, n=7) and normal human plasma (n=4). Ni-NTA bound proteins were analyzed by one-dimensional gel electrophoresis, followed by in-gel digestion for characterization using a LTQ-Orbitrap hybrid mass spectrometer. In addition, the loss of two non-hemoglobin related plasma proteins, thrombospondin1 (TSP1) and L-selectin, by Ni-NTA was determined by ELISA (SCD n=6, non-SCD controls n=2). Results Ni-NTA resulted in an average 60% decrease in plasma protein concentration, which was not hemolysis dependent. Specifically, Hb (7 peptides) and the top three proteins, alpha-2-macroglobulin (75 peptides), apolipoprotein B-100 (73 peptides), and albumin (42 peptides) were Ni-NTA bound. In addition, using an ELISA assay two non-hemoglobin associated plasma proteins TSP1 and L-selectin were decreased by Ni-NTA. Conclusions and clinical relevance Hb depletion with Ni-NTA is effective for Hb removal but is not specific. There is potential for deleterious depletion of potential biomarkers that may limit the applicability of this method. Consideration of alternate methods of Hb depletion for clinical proteomics may be warranted. PMID:21179892

  4. Development of a Listeria monocytogenes EGDe Partial Proteome Reference Map and Comparison with the Protein Profiles of Food Isolates

    PubMed Central

    Ramnath, Manilduth; Rechinger, K. Björn; Jänsch, Lothar; Hastings, John W.; Knøchel, Susanne; Gravesen, Anne

    2003-01-01

    A partially annotated proteome reference map of the food pathogen Listeria monocytogenes was developed for exponentially growing cells under standardized, optimal conditions by using the sequenced strain EGDe (serotype 1/2a) as a model organism. The map was developed by using a reproducible total protein extraction and two-dimensional (2-D) polyacrylamide gel electrophoresis analysis procedure, and it contained 33 identified proteins representing the four main protein functional classes. In order to facilitate analysis of membrane proteins, a protein compartmentalization procedure was assessed. The method used provided partial fractionation of membrane and cytosolic proteins. The total protein 2-D profiles of three serotype 1/2a strains and one serotype 1/2b strain isolated from food were compared to the L. monocytogenes EGDe proteome. An average of 13% of the major protein spots in the food strain proteomes were not matched in the strain EGDe proteome. The variation was greater for the less intense spots, and on average 28% of these spots were not matched. Two of the proteins identified in L. monocytogenes EGDe were missing in one or more of the food isolates. These two proteins were proteins involved in the main glycolytic pathway and in metabolism of coenzymes and prosthetic groups. The two corresponding genes were found by PCR amplification to be present in the four food isolates. Our results show that the L. monocytogenes EGDe reference map is a valuable starting point for analyses of strains having various origins and could be useful for analyzing the proteomes of different isolates of this pathogen. PMID:12788738

  5. Proteomic analysis of microvesicles from plasma of healthy donors reveals high individual variability.

    PubMed

    Bastos-Amador, Patricia; Royo, Felix; Gonzalez, Esperanza; Conde-Vancells, Javier; Palomo-Diez, Laura; Borras, Francesc E; Falcon-Perez, Juan M

    2012-06-27

    Healthy blood plasma is required for several therapeutic procedures. To maximize successful therapeutic outcomes it is critical to control the quality of blood plasma. Clearly initiatives to improve the safety of blood transfusions will have a high economical and social impact. A detailed knowledge of the composition of healthy blood plasma is essential to facilitate such improvements. Apart from free proteins, lipids and metabolites, blood plasma also contains cell-derived microvesicles, including exosomes and microparticles from several different cellular origins. In this study, we have purified microvesicles smaller than 220nm from plasma of healthy donors and performed proteomic, ultra-structural, biochemical and functional analyses. We have detected 161 microvesicle-associated proteins, including many associated with the complement and coagulation signal-transduction cascades. Several proteases and protease inhibitors associated with acute phase responses were present, indicating that these microvesicles may be involved in these processes. There was a remarkably high variability in the protein content of plasma from different donors. In addition, we report that this variability could be relevant for their interaction with cellular systems. This work provides valuable information on plasma microvesicles and a foundation to understand microvesicle biology and clinical implications.

  6. 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

    2016-04-13

    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.

  7. 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

  8. Plant plasma membrane protein extraction and solubilization for proteomic analysis.

    PubMed

    Santoni, Véronique

    2007-01-01

    The plasma membrane (PM) exists as the interface between the cytosol and the environment in all living cells and is one of the most complex and differentiated membrane. The identification and characterization of membrane proteins (either extrinsic or intrinsic) is a crucial challenge since many of these proteins are involved in essential cellular functions such as cell signaling, osmoregulation, nutrition, and metabolism. Methods to isolate PM fractions vary according to organisms, tissues, and cell type. This chapter emphasizes isolation, from the model plant Arabidopsis thaliana, of PM fractions from a microsomal membrane fraction by two-phase partitioning, a methodology that utilizes the different surface properties of membranes. PM proteins that do not span the lipid bilayer are generally well recovered after 2D gel electrophoresis. By contrast, the recovery of transmembrane proteins requires first the depletion of the PM fraction from soluble proteins, being either cytosolic contaminants or functionally associated proteins, and second, to the use of specific solubilization procedures. This chapter presents protocols to strip PM based on alkaline treatment of membranes and to solubilize hydrophobic proteins to increase their recovery on 2D gels. Aquaporins that are highly hydrophobic proteins are used to probe the relevance of the procedures.

  9. Placental Proteomics Provides Insights into Pathophysiology of Pre-Eclampsia and Predicts Possible Markers in Plasma.

    PubMed

    Mary, Sheon; Kulkarni, Mahesh J; Malakar, Dipankar; Joshi, Sadhana R; Mehendale, Savita S; Giri, Ashok P

    2017-02-03

    Pre-eclampsia is a hypertensive disorder characterized by the new onset of hypertension >140/90 mmHg and proteinuria after the 20th week of gestation. The disorder is multifactorial and originates with abnormal placentation. Comparison of the placental proteome of normotensive (n = 25) and pre-eclamptic (n = 25) patients by gel-free proteomic techniques identified a total of 2145 proteins in the placenta of which 180 were differentially expressed (>1.3 fold, p < 0.05). Gene ontology enrichment analysis of biological process suggested that the differentially expressed proteins belonged to various physiological processes such as angiogenesis, apoptosis, oxidative stress, hypoxia, and placental development, which are implicated in the pathophysiology of pre-eclampsia. Some of the differentially expressed proteins were monitored in the plasma by multiple reaction monitoring analysis, which showed an increase in apolipoproteins A-I and A-II in gestational weeks 26-30 (2-fold, p < 0.01), while haptoglobin and hemopexin decreased in gestational weeks 26-30 and week 40/at delivery (1.8 fold, p < 0.01) in pre-eclamptic patients. This study provides a proteomic insight into the pathophysiology of pre-eclampsia. Identified candidate proteins can be evaluated further for the development of potential biomarkers associated with pre-eclampsia pathogenesis.

  10. Proteomic analysis of plasma membrane and tonoplast from the leaves of mangrove plant Avicennia officinalis.

    PubMed

    Krishnamurthy, Pannaga; Tan, Xing Fei; Lim, Teck Kwang; Lim, Tit-Meng; Kumar, Prakash P; Loh, Chiang-Shiong; Lin, Qingsong

    2014-11-01

    In order to understand the salt tolerance and secretion in mangrove plant species, gel electrophoresis coupled with LC-MS-based proteomics was used to identify key transport proteins in the plasma membrane (PM) and tonoplast fractions of Avicennia officinalis leaves. PM and tonoplast proteins were purified using two-aqueous-phase partitioning and density gradient centrifugation, respectively. Forty of the 254 PM proteins and 31 of the 165 tonoplast proteins identified were predicted to have transmembrane domains. About 95% of the identified proteins could be classified based on their functions. The major classes of proteins were predicted to be involved in transport, metabolic processes, defense/stress response, and signal transduction, while a few of the proteins were predicted to be involved in other functions such as membrane trafficking. The main classes of transporter proteins identified included H(+) -ATPases, ATP-binding cassette transporters, and aquaporins, all of which could play a role in salt secretion. These data will serve as the baseline membrane proteomic dataset for Avicennia species. Further, this information can contribute to future studies on understanding the mechanism of salt tolerance in halophytes in addition to salt secretion in mangroves. All MS data have been deposited in the ProteomeXchange with identifier PXD000837 (http://proteomecentral.proteomexchange.org/dataset/PXD000837).

  11. Proteomic analysis of plasma from rats following total parenteral nutrition-induced liver injury.

    PubMed

    Tsai, Jai-Jen; Kuo, Hsing-Chun; Lee, Kam-Fai; Tsai, Tung-Hu

    2015-11-01

    Total parenteral nutrition (TPN) is provided as the primary nitrogen source to manage patients with intestinal failure who were not able to sustain themselves on enteral feeds. The most common complication of long-term TPN use is hepatitis. A proteomic approach was used to identify proteins that are differentially expressed in the plasma of rats following TPN-related acute liver injury. Six male rats were randomly assigned to either the saline infusion control group or the TPN infusion group. Our results demonstrate that TPN infusion in rats resulted in hepatic dysfunction and hepatocyte apoptosis. Five proteins that were differentially expressed between TPN infusion and normal rats were determined and validated in vivo. Fascinatingly, the proteomic differential displays, downregulated proteins included peroxiredoxin 2 (PRDX2), alpha-1-antiproteinase (A1AT), and fibrinogen gamma chain (FIBG), which were involved in oxidative stress, inflammatory respondence and cells apoptosis. After TPN infusion, two protein spots showed increased expression, namely, the glucagon receptor (GLR) protein and apolipoprotein A-1 (APOA1), which may mediate the effects of TPN administration on glycogen and lipid metabolism. In this study, proteomic analysis suggested TPN-related acute liver injury could be involved in limiting cellular protection mechanisms against oxidative stress-induced apoptosis. On the basis of the results, we also give molecular evidences replying TPN-related hepatitis.

  12. Plasma Proteomic Study in Pulmonary Arterial Hypertension Associated with Congenital Heart Diseases

    PubMed Central

    Zhang, Xi; Hou, Hai-Tao; Wang, Jun; Liu, Xiao-Cheng; Yang, Qin; He, Guo-Wei

    2016-01-01

    Pulmonary arterial hypertension associated with congenital heart disease (CHD-PAH) has serious consequence and plasma protein profiles in CHD-PAH are unknown. We aimed to reveal the differential plasma proteins in 272 CHD patients with or without PAH. Various types of CHD-PAH were studied. Differential plasma proteins were first detected by iTRAQ proteomic technology and those with significant clinical relevance were selected for further ELISA validation in new cohort of patients. Among the 190 differential plasma proteins detected by iTRAQ, carbamoyl-phosphate synthetase I (CPSI, related to urea cycle and endogenous nitric oxide production) and complement factor H-related protein 2 (CFHR2, related to complement system and coagulant mechanism) were selected for further ELISA validation in new cohort of 152 patients. Both CPSI and CFHR2 were down-regulated with decreased plasma levels (p < 0.01). Thus, we for the first time in CHD-PAH patients identified a large number of differential plasma proteins. The decreased CPSI expression in CHD-PAH patients may reveal a mechanism related to endogenous nitric oxide and the decrease of CFHR2 protein may demonstrate the deficiency of the immune system and coagulation mechanism. The findings may open a new direction for translational medicine in CHD-PAH with regard to the diagnosis and progress of the disease. PMID:27886187

  13. Plasma Proteomic Study in Pulmonary Arterial Hypertension Associated with Congenital Heart Diseases.

    PubMed

    Zhang, Xi; Hou, Hai-Tao; Wang, Jun; Liu, Xiao-Cheng; Yang, Qin; He, Guo-Wei

    2016-11-25

    Pulmonary arterial hypertension associated with congenital heart disease (CHD-PAH) has serious consequence and plasma protein profiles in CHD-PAH are unknown. We aimed to reveal the differential plasma proteins in 272 CHD patients with or without PAH. Various types of CHD-PAH were studied. Differential plasma proteins were first detected by iTRAQ proteomic technology and those with significant clinical relevance were selected for further ELISA validation in new cohort of patients. Among the 190 differential plasma proteins detected by iTRAQ, carbamoyl-phosphate synthetase I (CPSI, related to urea cycle and endogenous nitric oxide production) and complement factor H-related protein 2 (CFHR2, related to complement system and coagulant mechanism) were selected for further ELISA validation in new cohort of 152 patients. Both CPSI and CFHR2 were down-regulated with decreased plasma levels (p < 0.01). Thus, we for the first time in CHD-PAH patients identified a large number of differential plasma proteins. The decreased CPSI expression in CHD-PAH patients may reveal a mechanism related to endogenous nitric oxide and the decrease of CFHR2 protein may demonstrate the deficiency of the immune system and coagulation mechanism. The findings may open a new direction for translational medicine in CHD-PAH with regard to the diagnosis and progress of the disease.

  14. Plasma proteomic profiles from disease-discordant monozygotic twins suggest that molecular pathways are shared in multiple systemic autoimmune diseases*

    PubMed Central

    2011-01-01

    Introduction Although systemic autoimmune diseases (SAID) share many clinical and laboratory features, whether they also share some common features of pathogenesis remains unclear. We assessed plasma proteomic profiles among different SAID for evidence of common molecular pathways that could provide insights into pathogenic mechanisms shared by these diseases. Methods Differential quantitative proteomic analyses (one-dimensional reverse-phase liquid chromatography-mass spectrometry) were performed to assess patterns of plasma protein expression. Monozygotic twins (four pairs discordant for systemic lupus erythematosus, four pairs discordant for juvenile idiopathic arthritis and two pairs discordant for juvenile dermatomyositis) were studied to minimize polymorphic gene effects. Comparisons were also made to 10 unrelated, matched controls. Results Multiple plasma proteins, including acute phase reactants, structural proteins, immune response proteins, coagulation and transcriptional factors, were differentially expressed similarly among the different SAID studied. Multivariate Random Forest modeling identified seven proteins whose combined altered expression levels effectively segregated affected vs. unaffected twins. Among these seven proteins, four were also identified in univariate analyses of proteomic data (syntaxin 17, α-glucosidase, paraoxonase 1, and the sixth component of complement). Molecular pathway modeling indicated that these factors may be integrated through interactions with a candidate plasma biomarker, PON1 and the pro-inflammatory cytokine IL-6. Conclusions Together, these data suggest that different SAID may share common alterations of plasma protein expression and molecular pathways. An understanding of the mechanisms leading to the altered plasma proteomes common among these SAID may provide useful insights into their pathogeneses. PMID:22044644

  15. An efficient organic solvent based extraction method for the proteomic analysis of Arabidopsis plasma membranes.

    PubMed

    Mitra, Srijeet K; Walters, Benjamin T; Clouse, Steven D; Goshe, Michael B

    2009-06-01

    Membrane proteins are involved in diverse cellular processes and are an integral component of many signaling cascades, but due to their highly hydrophobic nature and the complexities associated with studying these proteins in planta, alternative methods are being developed to better characterize these proteins on a proteome-wide scale. In our previous work ( Mitra , S. K. et al. J. Proteome Res. 2007 , 6 , ( 5 ), 1933 - 50 ), methanol-assisted solubilization was determined to facilitate the identification of both hydrophobic and hydrophilic membrane proteins compared to Brij-58 solubilization and was particularly effective for leucine-rich repeat receptor-like kinases (LRR RLKs). To improve peptide identification and to overcome sample losses after tryptic digestion, we have developed an effective chloroform extraction method to promote plasma membrane protein identification. The use of chloroform extraction over traditional solid-phase extraction (SPE) prior to off-line strong cation exchange liquid chromatography (SCXC) and reversed-phase liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis facilitated the removal of chlorophylls, major contaminants of plant tissue preparations that can affect downstream analysis, in addition to the effective removal of trypsin used in the digestion. On the basis of a statistically derived 5% false discovery rate, the chloroform extraction procedure increased the identification of unique peptides for plasma membrane proteins over SPE by 70% which produced nearly a 2-fold increase in detection of membrane transporters and LRR RLKs without increased identification of contaminating Rubisco and ribosomal peptides. Overall, the combined use of methanol and chloroform provides an effective method to study membrane proteins and can be readily applied to other tissues and cells types for proteomic analysis.

  16. Effects of Increased CO2 on Fish Gill and Plasma Proteome

    PubMed Central

    Bresolin de Souza, Karine; Jutfelt, Fredrik; Kling, Peter; Förlin, Lars; Sturve, Joachim

    2014-01-01

    Ocean acidification and warming are both primarily caused by increased levels of atmospheric CO2, and marine organisms are exposed to these two stressors simultaneously. Although the effects of temperature on fish have been investigated over the last century, the long-term effects of moderate CO2 exposure and the combination of both stressors are almost entirely unknown. A proteomics approach was used to assess the adverse physiological and biochemical changes that may occur from the exposure to these two environmental stressors. We analysed gills and blood plasma of Atlantic halibut (Hippoglossus hippoglossus) exposed to temperatures of 12°C (control) and 18°C (impaired growth) in combination with control (400 µatm) or high-CO2 water (1000 µatm) for 14 weeks. The proteomic analysis was performed using two-dimensional gel electrophoresis (2DE) followed by Nanoflow LC-MS/MS using a LTQ-Orbitrap. The high-CO2 treatment induced the up-regulation of immune system-related proteins, as indicated by the up-regulation of the plasma proteins complement component C3 and fibrinogen β chain precursor in both temperature treatments. Changes in gill proteome in the high-CO2 (18°C) group were mostly related to increased energy metabolism proteins (ATP synthase, malate dehydrogenase, malate dehydrogenase thermostable, and fructose-1,6-bisphosphate aldolase), possibly coupled to a higher energy demand. Gills from fish exposed to high-CO2 at both temperature treatments showed changes in proteins associated with increased cellular turnover and apoptosis signalling (annexin 5, eukaryotic translation elongation factor 1γ, receptor for protein kinase C, and putative ribosomal protein S27). This study indicates that moderate CO2-driven acidification, alone and combined with high temperature, can elicit biochemical changes that may affect fish health. PMID:25058324

  17. 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.

  18. A Plasma Proteomic Approach in Rett Syndrome: Classical versus Preserved Speech Variant

    PubMed Central

    Cortelazzo, Alessio; Guerranti, Roberto; De Felice, Claudio; Leoncini, Silvia; Landi, Claudia; Montomoli, Barbara; Sticozzi, Claudia; Ciccoli, Lucia; Hayek, Joussef

    2013-01-01

    Rett syndrome (RTT) is a progressive neurodevelopmental disorder mainly caused by mutations in the gene encoding the methyl-CpG-binding protein 2 (MeCP2). Although over 200 mutations types have been identified so far, nine of which the most frequent ones. A wide phenotypical heterogeneity is a well-known feature of the disease, with different clinical presentations, including the classical form and the preserved speech variant (PSV). Aim of the study was to unveil possible relationships between plasma proteome and phenotypic expression in two cases of familial RTT represented by two pairs of sisters, harbor the same MECP2 gene mutation while being dramatically discrepant in phenotype, that is, classical RTT versus PSV. Plasma proteome was analysed by 2-DE/MALDI-TOF MS. A significant overexpression of six proteins in the classical sisters was detected as compared to the PSV siblings. A total of five out of six (i.e., 83.3%) of the overexpressed proteins were well-known acute phase response (APR) proteins, including alpha-1-microglobulin, haptoglobin, fibrinogen beta chain, alpha-1-antitrypsin, and complement C3. Therefore, the examined RTT siblings pairs proved to be an important benchmark model to test the molecular basis of phenotypical expression variability and to identify potential therapeutic targets of the disease. PMID:24453418

  19. Plasma proteomic analysis of stable coronary artery disease indicates impairment of reverse cholesterol pathway

    PubMed Central

    Basak, Trayambak; Tanwar, Vinay Singh; Bhardwaj, Gourav; Bhardwaj, Nitin; Ahmad, Shadab; Garg, Gaurav; V, Sreenivas; Karthikeyan, Ganesan; Seth, Sandeep; Sengupta, Shantanu

    2016-01-01

    Coronary artery disease (CAD) is one of the largest causes of death worldwide yet the traditional risk factors, although useful in identifying people at high risk, lack the desired predictive accuracy. Techniques like quantitative plasma proteomics holds immense potential to identify newer markers and this study (conducted in three phases) was aimed to identify differentially expressed proteins in stable CAD patients. In the first (discovery) phase, plasma from CAD cases (angiographically proven) and controls were subjected to iTRAQ based proteomic analysis. Proteins found to be differentially expressed were then validated in the second and third (verification and validation) phases in larger number of (n = 546) samples. After multivariate logistic regression adjusting for confounding factors (age, diet, etc.), four proteins involved in the reverse cholesterol pathway (Apo A1, ApoA4, Apo C1 and albumin) along with diabetes and hypertension were found to be significantly associated with CAD and could account for approximately 88% of the cases as revealed by ROC analysis. The maximum odds ratio was found to be 6.70 for albumin (p < 0.0001), followed by Apo AI (5.07, p < 0.0001), Apo CI (4.03, p = 0.001), and Apo AIV (2.63, p = 0.003). Down-regulation of apolipoproteins and albumin implicates the impairment of reverse cholesterol pathway in CAD. PMID:27350024

  20. Plasma proteomic analysis of stable coronary artery disease indicates impairment of reverse cholesterol pathway.

    PubMed

    Basak, Trayambak; Tanwar, Vinay Singh; Bhardwaj, Gourav; Bhardwaj, Nitin; Ahmad, Shadab; Garg, Gaurav; V, Sreenivas; Karthikeyan, Ganesan; Seth, Sandeep; Sengupta, Shantanu

    2016-06-28

    Coronary artery disease (CAD) is one of the largest causes of death worldwide yet the traditional risk factors, although useful in identifying people at high risk, lack the desired predictive accuracy. Techniques like quantitative plasma proteomics holds immense potential to identify newer markers and this study (conducted in three phases) was aimed to identify differentially expressed proteins in stable CAD patients. In the first (discovery) phase, plasma from CAD cases (angiographically proven) and controls were subjected to iTRAQ based proteomic analysis. Proteins found to be differentially expressed were then validated in the second and third (verification and validation) phases in larger number of (n = 546) samples. After multivariate logistic regression adjusting for confounding factors (age, diet, etc.), four proteins involved in the reverse cholesterol pathway (Apo A1, ApoA4, Apo C1 and albumin) along with diabetes and hypertension were found to be significantly associated with CAD and could account for approximately 88% of the cases as revealed by ROC analysis. The maximum odds ratio was found to be 6.70 for albumin (p < 0.0001), followed by Apo AI (5.07, p < 0.0001), Apo CI (4.03, p = 0.001), and Apo AIV (2.63, p = 0.003). Down-regulation of apolipoproteins and albumin implicates the impairment of reverse cholesterol pathway in CAD.

  1. A plasma proteomic approach in Rett syndrome: classical versus preserved speech variant.

    PubMed

    Cortelazzo, Alessio; Guerranti, Roberto; De Felice, Claudio; Signorini, Cinzia; Leoncini, Silvia; Pecorelli, Alessandra; Landi, Claudia; Bini, Luca; Montomoli, Barbara; Sticozzi, Claudia; Ciccoli, Lucia; Valacchi, Giuseppe; Hayek, Joussef

    2013-01-01

    Rett syndrome (RTT) is a progressive neurodevelopmental disorder mainly caused by mutations in the gene encoding the methyl-CpG-binding protein 2 (MeCP2). Although over 200 mutations types have been identified so far, nine of which the most frequent ones. A wide phenotypical heterogeneity is a well-known feature of the disease, with different clinical presentations, including the classical form and the preserved speech variant (PSV). Aim of the study was to unveil possible relationships between plasma proteome and phenotypic expression in two cases of familial RTT represented by two pairs of sisters, harbor the same MECP2 gene mutation while being dramatically discrepant in phenotype, that is, classical RTT versus PSV. Plasma proteome was analysed by 2-DE/MALDI-TOF MS. A significant overexpression of six proteins in the classical sisters was detected as compared to the PSV siblings. A total of five out of six (i.e., 83.3%) of the overexpressed proteins were well-known acute phase response (APR) proteins, including alpha-1-microglobulin, haptoglobin, fibrinogen beta chain, alpha-1-antitrypsin, and complement C3. Therefore, the examined RTT siblings pairs proved to be an important benchmark model to test the molecular basis of phenotypical expression variability and to identify potential therapeutic targets of the disease.

  2. Affinity proteomics discovers decreased levels of AMFR in plasma from Osteoporosis patients

    PubMed Central

    Qundos, Ulrika; Drobin, Kimi; Mattsson, Cecilia; Hong, Mun‐Gwan; Sjöberg, Ronald; Forsström, Björn; Solomon, David; Uhlén, Mathias; Nilsson, Peter; Michaëlsson, Karl

    2015-01-01

    Purpose Affinity proteomic approaches by antibody bead arrays enable multiplexed analysis of proteins in body fluids. In the presented study, we investigated blood plasma within osteoporosis to discovery differential protein profiles and to propose novel biomarkers candidates for subsequent studies. Experimental design Starting with 4608 antibodies and plasma samples from 22 women for an untargeted screening, a set of 72 proteins were suggested for further analysis. Complementing these with targets from literature and other studies, a targeted bead array of 180 antibodies was built to profile for 92 proteins in plasma samples of 180 women from two independent population‐based studies. Results Differential profiles between osteoporosis patients and matched controls were discovered for 12 proteins in at least one of the two study sets. Among these targets, the levels of autocrine motility factor receptor (AMFR) were concordantly lower in plasma of female osteoporosis patients. Subsequently, verification of anti‐AMFR antibody selectivity was conducted using high‐density peptide and protein arrays, and Western blotting. Conclusions and clinical relevance Further validation in additional study sets will be needed to determine the clinical value of the observed decrease in AMFR plasma levels in osteoporosis patients, but AMFR may aid our understanding of disease mechanisms and could support existing tools for diagnosis and monitoring of patient mobility within osteoporosis. PMID:25689831

  3. Proteomic analysis of plasma membrane and secretory vesicles from human neutrophils

    PubMed Central

    Jethwaney, Deepa; Islam, Md Rafiqul; Leidal, Kevin G; de Bernabe, Daniel Beltran-Valero; Campbell, Kevin P; Nauseef, William M; Gibson, Bradford W

    2007-01-01

    Background Polymorphonuclear neutrophils (PMN) constitute an essential cellular component of innate host defense against microbial invasion and exhibit a wide array of responses both to particulate and soluble stimuli. As the cells recruited earliest during acute inflammation, PMN respond rapidly and release a variety of potent cytotoxic agents within minutes of exposure to microbes or their products. PMN rely on the redistribution of functionally important proteins, from intracellular compartments to the plasma membrane and phagosome, as the means by which to respond quickly. To determine the range of membrane proteins available for rapid recruitment during PMN activation, we analyzed the proteins in subcellular fractions enriched for plasma membrane and secretory vesicles recovered from the light membrane fraction of resting PMN after Percoll gradient centrifugation and free-flow electrophoresis purification using mass spectrometry-based proteomics methods. Results To identify the proteins light membrane fractions enriched for plasma membrane vesicles and secretory vesicles, we employed a proteomic approach, first using MALDI-TOF (peptide mass fingerprinting) and then by HPLC-MS/MS using a 3D ion trap mass spectrometer to analyze the two vesicle populations from resting PMN. We identified several proteins that are functionally important but had not previously been recovered in PMN secretory vesicles. Two such proteins, 5-lipoxygenase-activating protein (FLAP) and dysferlin were further validated by immunoblot analysis. Conclusion Our data demonstrate the broad array of proteins present in secretory vesicles that provides the PMN with the capacity for remarkable and rapid reorganization of its plasma membrane after exposure to proinflammatory agents or stimuli. PMID:17692124

  4. Immunodepletion Plasma Proteomics by TripleTOF 5600 and Orbitrap Elite/LTQ-Orbitrap Velos/Q Exactive Mass Spectrometers

    PubMed Central

    Patel, Bhavinkumar B.; Kelsen, Steven G.; Braverman, Alan; Swinton, Derrick J.; Gafken, Philip R.; Jones, Lisa A.; Lane, William S.; Neveu, John M.; Leung, Hon-Chiu E.; Shaffer, Scott A.; Leszyk, John D.; Stanley, Bruce A.; Fox, Todd E.; Stanley, Anne; Hall, Michael J.; Hampel, Heather; South, Christopher D.; de la Chapelle, Albert; Burt, Randall W.; Jones, David A.; Kopelovich, Levy; Yeung, Anthony T.

    2013-01-01

    Plasma proteomic experiments performed rapidly and economically using several of the latest high-resolution mass spectrometers were compared. Four quantitative hyperfractionated plasma proteomics experiments were analyzed in replicates by two AB SCIEX TripleTOF 5600 and three Thermo Scientific Orbitrap (Elite/LTQ-Orbitrap Velos/Q Exactive) instruments. Each experiment compared two iTRAQ isobaric-labeled immunodepleted plasma proteomes, provided as 30 labeled peptide fractions. 480 LC-MS/MS runs delivered >250 GB of data in two months. Several analysis algorithms were compared. At 1 % false discovery rate, the relative comparative findings concluded that the Thermo Scientific Q Exactive Mass Spectrometer resulted in the highest number of identified proteins and unique sequences with iTRAQ quantitation. The confidence of iTRAQ fold-change for each protein is dependent on the overall ion statistics (Mascot Protein Score) attainable by each instrument. The benchmarking also suggested how to further improve the mass spectrometry parameters and HPLC conditions. Our findings highlight the special challenges presented by the low abundance peptide ions of iTRAQ plasma proteome because the dynamic range of plasma protein abundance is uniquely high compared with cell lysates, necessitating high instrument sensitivity. PMID:24004147

  5. Mining the human plasma proteome with three-dimensional strategies by high-resolution Quadrupole Orbitrap Mass Spectrometry.

    PubMed

    Zhao, Yan; Chang, Cheng; Qin, Peibin; Cao, Qichen; Tian, Fang; Jiang, Jing; Li, Xianyu; Yu, Wenfeng; Zhu, Yunping; He, Fuchu; Ying, Wantao; Qian, Xiaohong

    2016-01-21

    Human plasma is a readily available clinical sample that reflects the status of the body in normal physiological and disease states. Although the wide dynamic range and immense complexity of plasma proteins are obstacles, comprehensive proteomic analysis of human plasma is necessary for biomarker discovery and further verification. Various methods such as immunodepletion, protein equalization and hyper fractionation have been applied to reduce the influence of high-abundance proteins (HAPs) and to reduce the high level of complexity. However, the depth at which the human plasma proteome has been explored in a relatively short time frame has been limited, which impedes the transfer of proteomic techniques to clinical research. Development of an optimal strategy is expected to improve the efficiency of human plasma proteome profiling. Here, five three-dimensional strategies combining HAP depletion (the 1st dimension) and protein fractionation (the 2nd dimension), followed by LC-MS/MS analysis (the 3rd dimension) were developed and compared for human plasma proteome profiling. Pros and cons of the five strategies are discussed for two issues: HAP depletion and complexity reduction. Strategies A and B used proteome equalization and tandem Seppro IgY14 immunodepletion, respectively, as the first dimension. Proteome equalization (strategy A) was biased toward the enrichment of basic and low-molecular weight proteins and had limited ability to enrich low-abundance proteins. By tandem removal of HAPs (strategy B), the efficiency of HAP depletion was significantly increased, whereas more off-target proteins were subtracted simultaneously. In the comparison of complexity reduction, strategy D involved a deglycosylation step before high-pH RPLC separation. However, the increase in sequence coverage did not increase the protein number as expected. Strategy E introduced SDS-PAGE separation of proteins, and the results showed oversampling of HAPs and identification of fewer

  6. Proteome Reference Maps of Vegetative Tissues in Pea. An Investigation of Nitrogen Mobilization from Leaves during Seed Filling1

    PubMed Central

    Schiltz, Séverine; Gallardo, Karine; Huart, Myriam; Negroni, Luc; Sommerer, Nicolas; Burstin, Judith

    2004-01-01

    A proteomic approach was used to analyze protein changes during nitrogen mobilization (N mobilization) from leaves to filling seeds in pea (Pisum sativum). First, proteome reference maps were established for mature leaves and stems. They displayed around 190 Coomassie Blue-stained spots with pIs from 4 to 7. A total of 130 spots were identified by mass spectrometry as corresponding to 80 different proteins implicated in a variety of cellular functions. Although the leaf proteome map contained more abundant spots, corresponding to proteins involved in energy/carbon metabolism, than the stem map, their comparison revealed a highly similar protein profile. Second, the leaf proteome map was used to analyze quantitative variations in leaf proteins during N mobilization. Forty percent of the spots showed significant changes in their relative abundance in the total protein extract. The results confirmed the importance of Rubisco as a source of mobilizable nitrogen, and suggested that in pea leaves the rate of degradation of Rubisco may vary throughout N mobilization. Correlated with the loss of Rubisco was an increase in relative abundance of chloroplastic protease regulatory subunits. Concomitantly, the relative abundance of some proteins related to the photosynthetic apparatus (Rubisco activase, Rubisco-binding proteins) and of several chaperones increased. A role for these proteins in the maintenance of a Rubisco activation state and in the PSII repair during the intense proteolytic activity within the chloroplasts was proposed. Finally, two 14-3-3-like proteins, with a potential regulatory role, displayed differential expression patterns during the massive remobilization of nitrogen. PMID:15299134

  7. Proteome reference maps of vegetative tissues in pea. An investigation of nitrogen mobilization from leaves during seed filling.

    PubMed

    Schiltz, Séverine; Gallardo, Karine; Huart, Myriam; Negroni, Luc; Sommerer, Nicolas; Burstin, Judith

    2004-08-01

    A proteomic approach was used to analyze protein changes during nitrogen mobilization (N mobilization) from leaves to filling seeds in pea (Pisum sativum). First, proteome reference maps were established for mature leaves and stems. They displayed around 190 Coomassie Blue-stained spots with pIs from 4 to 7. A total of 130 spots were identified by mass spectrometry as corresponding to 80 different proteins implicated in a variety of cellular functions. Although the leaf proteome map contained more abundant spots, corresponding to proteins involved in energy/carbon metabolism, than the stem map, their comparison revealed a highly similar protein profile. Second, the leaf proteome map was used to analyze quantitative variations in leaf proteins during N mobilization. Forty percent of the spots showed significant changes in their relative abundance in the total protein extract. The results confirmed the importance of Rubisco as a source of mobilizable nitrogen, and suggested that in pea leaves the rate of degradation of Rubisco may vary throughout N mobilization. Correlated with the loss of Rubisco was an increase in relative abundance of chloroplastic protease regulatory subunits. Concomitantly, the relative abundance of some proteins related to the photosynthetic apparatus (Rubisco activase, Rubisco-binding proteins) and of several chaperones increased. A role for these proteins in the maintenance of a Rubisco activation state and in the PSII repair during the intense proteolytic activity within the chloroplasts was proposed. Finally, two 14-3-3-like proteins, with a potential regulatory role, displayed differential expression patterns during the massive remobilization of nitrogen.

  8. Proteomic Analysis of the Ontogenetic Variability in Plasma Composition of Juvenile and Adult Bothrops jararaca Snakes

    PubMed Central

    de Morais-Zani, Karen; Grego, Kathleen Fernandes; Tanaka, Aparecida Sadae; Tanaka-Azevedo, Anita Mitico

    2013-01-01

    The ontogenetic variability in venom composition of some snake genera, including Bothrops, as well as the biological implications of such variability and the search of new molecules that can neutralize the toxic components of these venoms have been the subject of many studies. Thus, considering the resistance of Bothrops jararaca to the toxic action of its own venom and the ontogenetic variability in venom composition described in this species, a comparative study of the plasma composition of juvenile and adult B. jararaca snakes was performed through a proteomic approach based on 2D electrophoresis and mass spectrometry, which allowed the identification of proteins that might be present at different levels during ontogenetic development. Among the proteins identified by mass spectrometry, antihemorrhagic factor Bj46a was found only in adult plasma. Moreover, two spots identified as phospholipase A2 inhibitors were significantly increased in juvenile plasma, which can be related to the higher catalytic PLA2 activity shown by juvenile venom in comparison to that of adult snakes. This work shows the ontogenetic variability of B. jararaca plasma, and that these changes can be related to the ontogenetic variability described in its venom. PMID:24062950

  9. Proteomic profiling of human plasma exosomes identifies PPAR{gamma} as an exosome-associated protein

    SciTech Connect

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

    2009-01-16

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

  10. Comprehensive Analysis of Low-Molecular-Weight Human Plasma Proteome Using Top-Down Mass Spectrometry.

    PubMed

    Cheon, Dong Huey; Nam, Eun Ji; Park, Kyu Hyung; Woo, Se Joon; Lee, Hye Jin; Kim, Hee Cheol; Yang, Eun Gyeong; Lee, Cheolju; Lee, Ji Eun

    2016-01-04

    While human plasma serves as a great source for disease diagnosis, low-molecular-weight (LMW) proteome (<30 kDa) has been shown to contain a rich source of diagnostic biomarkers. Here we employ top-down mass spectrometry to analyze the LMW proteoforms present in four types of human plasma samples pooled from three healthy controls (HCs) without immunoaffinity depletion and with depletion of the top two, six, and seven high-abundance proteins. The LMW proteoforms were first fractionated based on molecular weight using gel-eluted liquid fraction entrapment electrophoresis (GELFrEE). Then, the GELFrEE fractions containing up to 30 kDa were subjected to nanocapillary-LC-MS/MS, and the high-resolution MS and MS/MS data were processed using ProSightPC 3.0. As a result, a total of 442 LMW proteins and cleaved products, including those with post-translational modifications and single amino acid variations, were identified. From additional comparative analysis of plasma samples without immunoaffinity depletion between HCs and colorectal cancer (CRC) patients via top-down approach, tens of LMW proteoforms, including platelet factor 4, were found to show >1.5-fold changes between the plasma samples of HCs and CRC patients, and six of the LMW proteins were verified by Western blot analysis.

  11. Plasma biochemistry reference values of wild bonnethead sharks, Sphyrna tiburo.

    PubMed

    Harms, Craig; Ross, Trisha; Segars, Al

    2002-01-01

    Elasmobranchs (sharks, skates, and rays) are of commercial, sport, research, and exhibit importance, however, blood chemistry reference values have been determined for few of these species. The purpose of this study was to establish plasma biochemistry and PCV reference values for wild bonnethead sharks (Sphyrna tiburo). Heparinized blood samples were collected from 24 bonnethead sharks at the time of capture in trawl nets off the coast of South Carolina and Georgia. Weight, length, PCV, total solids (TS, by refractometry), and plasma biochemical analyses were done using standard techniques. Wilcoxon rank-sum and Kendall tau b tests were used to compare values by animal size, boat and sex; 1-way ANOVA was used to compare TS and total protein (TP) concentrations. Median (quartile; minimum-maximum) values were as follows: PCV 22% (22%, 26%; 17-28%), TS 6.3 (6.0, 6.8; 5.8-7.5) g/dL, total protein 2.9 (2.7, 3.4; 2.2-4.3) g/dL, albumin 0.4 (0.4, 0.4; 0.3-0.5) g/dL, globulins 2.6 (2.3, 3.0;1.9-3.8) g/dL, sodium 282 (279, 285;273-292) mmol/L, potassium 7.3 (6.4, 7.9; 5.7-9.2) mmol/L, chloride 290 (285, 296; 277-304) mmol/L, total CO2 3 (2, 4; 0-5) mmol/L, calcium 16.8 (16.2, 17.4;15.8-18.2) mg/dL, phosphorus 8.8 (7.5, 10.0; 5.9-12.7) mg/dL, urea nitrogen 1004 (986, 1028; 944-1068) mg/dL, creatinine <0.1 mg/dL, glucose 184 (165, 191; 155-218) mg/dL, aspartate aminotransferase 42 (33, 66; 15-132) U/L, lactate dehydrogenase <5 U/L, creatine kinase 82 (47, 233; 18-725) U/L, and osmolality 1094 (1078, 1111; 1056-1139) mOsm/kg. No differences based on sex were detected. TS and TP values were related by the fitted line TS = (1.006 X TP) + 3.318. Values reported here will be useful for evaluating the health status of bonnetheads in wild and captive research conditions and in exhibits.

  12. Proteomic characterization of freeze-dried human plasma: providing treatment of bleeding disorders without the need for a cold chain.

    PubMed

    Steil, Leif; Thiele, Thomas; Hammer, Elke; Bux, Jürgen; Kalus, Monika; Völker, Uwe; Greinacher, Andreas

    2008-11-01

    Transfusion of human plasma is a basic treatment for severe coagulopathies, especially in major bleeding. The required logistics to provide plasma is challenging because of the need to maintain a cold chain. This disadvantage could be overcome by lyophilized plasma. However, it is unknown to what extent lyophilization alters plasma proteins. Quantitative proteomic technologies were applied to monitor protein changes during production of lyophilized, solvent/detergent (S/D)-treated plasma. The impact of S/D treatment and lyophilization on the plasma proteome was evaluated by differential in-gel electrophoresis (2D-DIGE), and proteins were characterized by mass spectrometry. Clotting factor activities were determined in lyophilized S/D-treated plasma after 24 months of storage at room temperature. By 2D-DIGE, 600 individual protein spots were compared. Lyophilization did not change any of the 600 spots, whereas pathogen inactivation caused significant changes of 38 spots including alpha1-antitrypsin, alpha1-antichymotrypsin, and alpha2-antiplasmin. Clotting factor activities remained stable over 24 months of storage. Lyophilization of human plasma neither alters its protein composition nor impairs its clotting capacity. It does not require cost-intensive logistics for storage and transport and can be quickly reconstituted. It is suggested that lyophilized, pathogen-inactivated plasma is an attractive option to provide the most important basic treatment for severe coagulopathies in areas without cold chain and to provide plasma with reduced time delay in emergency situations.

  13. Plasma membrane proteomics of human embryonic stem cells and human embryonal carcinoma cells.

    PubMed

    Dormeyer, Wilma; van Hoof, Dennis; Braam, Stefan R; Heck, Albert J R; Mummery, Christine L; Krijgsveld, Jeroen

    2008-07-01

    Human embryonic stem cells (hESCs) are of immense interest in regenerative medicine as they can self-renew indefinitely and can give rise to any adult cell type. Human embryonal carcinoma cells (hECCs) are the malignant counterparts of hESCs found in testis tumors. hESCs that have acquired chromosomal abnormalities in culture are essentially indistinguishable from hECC. Direct comparison of karyotypically normal hESCs with hECCs could lead to understanding differences between their mechanisms of growth control and contribute to implementing safe therapeutic use of stem cells without the development of germ cell cancer. While several comparisons of hECCs and hESCs have been reported, their cell surface proteomes are largely unknown, partly because plasma membrane proteomics is still a major challenge. Here, we present a strategy for the identification of plasma membrane proteins that has been optimized for application to the relatively small numbers of stem cells normally available, and that does not require tedious cell fractionation. The method led to the identification of 237 and 219 specific plasma membrane proteins in the hESC line HUES-7 and the hECC line NT2/D1, respectively. In addition to known stemness-associated cell surface markers like ALP, CD9, and CTNNB, a large number of receptors, transporters, signal transducers, and cell-cell adhesion proteins were identified. Our study revealed that several Hedgehog and Wnt pathway members are differentially expressed in hESCs and hECCs including NPC1, FZD2, FZD6, FZD7, LRP6, and SEMA4D, which play a pivotal role in stem cell self-renewal and cancer growth. Various proteins encoded on chromosome 12p, duplicated in testicular cancer, were uniquely identified in hECCs. These included GAPDH, LDHB, YARS2, CLSTN3, CSDA, LRP6, NDUFA9, and NOL1, which are known to be upregulated in testicular cancer. Distinct HLA molecules were revealed on the surface of hESCs and hECCs, despite their low abundance. Results were

  14. A high content in lipid-modified peripheral proteins and integral receptor kinases features in the arabidopsis plasma membrane proteome.

    PubMed

    Marmagne, Anne; Ferro, Myriam; Meinnel, Thierry; Bruley, Christophe; Kuhn, Lauriane; Garin, Jérome; Barbier-Brygoo, Hélène; Ephritikhine, Geneviève

    2007-11-01

    The proteomics of plasma membrane has brought to date only scarce and partial information on the actual protein repertoire. In this work, the plant plasma membrane proteome of Arabidopsis thaliana was investigated. A highly purified plasma membrane fraction was washed by NaCl and Na2CO3 salts, and the insoluble fractions were further analyzed by nano-LC-MS/MS. With 446 proteins identified, we hereby describe the largest plasma membrane proteome diversity reported so far. Half of the proteins were predicted to display transmembrane domains and/or to be anchored to the membrane, validating a posteriori the pertinence of the approach. A fine analysis highlighted two main specific and novel features. First, the main functional category is represented by a majority of as yet unreported signaling proteins, including 11% receptor-like kinases. Second, 16% of the identified proteins are predicted to be lipid-modified, specifically involving double lipid linkage through N-terminal myristoylation, S-palmitoylation, C-terminal prenylation, or glycosylphosphatidylinositol anchors. Thus, our approach led for the first time to the identification of a large number of peripheral proteins as part of the plasma membrane and allowed the functionality of the plasma membrane in the cell context to be reconsidered.

  15. Fabry disease: characterisation of the plasma proteome pre- and post-enzyme replacement therapy.

    PubMed

    Heo, Sun Hee; Kang, Eungu; Kim, Yoon-Myung; Go, Heounjeong; Kim, Kyung Yong; Jung, Jae Yong; Kang, Minji; Kim, Gu-Hwan; Kim, Jae-Min; Choi, In-Hee; Choi, Jin-Ho; Jung, Sung-Chul; Desnick, Robert J; Yoo, Han-Wook; Lee, Beom Hee

    2017-08-23

    Fabry disease is characterised by the progressive accumulation of globotriaosylceramide (Gb3) and related glycosphingolipids in vascular endothelial cells. Enzyme replacement therapy (ERT) clears this accumulation. We analysed plasma proteome profiles before and after ERT to characterise its molecular pathology. Two-dimensional electrophoresis and matrix-assisted laser desorption/ionisation-time of flight tandem mass spectrometry (MALDI-TOF MS) and tandem mass spectrometry (MS/MS) were done using plasma samples before and after ERT in eight patients with classical Fabry disease RESULTS: After short-term ERT (4-12 months), the levels of 15 plasma proteins involved in inflammation, oxidative and ischaemic injury, or complement activation were reduced significantly. Among them, β-actin (ACTB), inactivated complement C3b (iC3b), and C4B were elevated significantly in pre-ERT Fabry disease plasma compared with control plasma. After longer-term ERT (46-96 months), iC3b levels gradually decreased, whereas the levels of other proteins varied. The gradual reduction of iC3b was comparable to that of Gb3 levels. In addition, iC3b increased significantly in pre-ERT Fabry disease mouse plasma, and C3 deposits were notable in renal tissues of pre-enzyme replacement therapy patients. These results indicated that C3-mediated complement activation might be altered in Fabry disease and ERT might promote its stabilisation. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  16. Effect of simulated air dive and decompression sickness on the plasma proteome of rats.

    PubMed

    Lautridou, Jacky; Pichereau, Vianney; Artigaud, Sébastien; Buzzacott, Peter; Wang, Qiong; Bernay, Benoit; Driad, Sabrina; Mazur, Aleksandra; Lambrechts, Kate; Théron, Michael; Guerrero, François

    2016-05-01

    Decompression sickness (DCS) is a poorly understood systemic disease caused by inadequate desaturation following a reduction in ambient pressure. Although recent studies highlight the importance of circulating factors, the available data are still puzzling. In this study, we aimed to identify proteins and biological pathways involved in the development of DCS in rats. Eighteen male Sprague-Dawley rats were subjected to a same simulated air dive to 1000 kPa absolute pressure and divided into two groups: no DCS or DCS. A third control group remained at atmospheric pressure. Venous blood was collected after hyperbaric exposure and the plasma proteomes from four individuals per group were analyzed by using a two-dimensional electrophoresis-based proteomic strategy. Quantitative analysis identified nine protein spots with abundances significantly changed (false discovery rate < 0.1) between the tested conditions. Three protein spots, identified as Apolipoprotein A1, Serine Protease Inhibitor A3K (Serpin A3K), and Alpha-1-antiproteinase, appeared increased in DCS animals but displayed only weak changes. By contrast, one protein spot identified as Transthyretin (TTR) dramatically decreased (i.e. quite disappeared) in animals displaying DCS symptoms. Before diving, TTR level was not different in DCS than nondiving group. These results may lead to the use of TTR as an early biomarker of DCS. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Diving into the rat plasma proteome to get to the bottom of decompression sickness.

    PubMed

    Eftedal, Ingrid

    2016-07-01

    Decompression sickness (DCS) is the collective term for an array of signs and symptoms triggered by ambient pressure reduction. It is of particular concern to divers as they decompress on ascend from depth to sea surface, but despite a long history of studies the determinants of DCS risk are incompletely understood and there are no validated biomarkers. In this issue of Proteomics Clinical Applications, Lautridou et al. [8] report on their search for DCS biomarkers in rats exposed to simulated diving. By comparing the plasma proteomes from animals showing neurological symptoms to those emerging from dives unaffected, they identified several high-abundance proteins not previously associated with DCS. The most significant finding was a near depletion of thyroxine- and vitamin A transporter transthyretin in symptomatic rats. In addition to their potential role as diagnostic biomarkers, the proteins identified in Lautridou's study may offer new pieces in the yet incomplete puzzle of DCS etiology. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. The plasma membrane proteome of maize roots grown under low and high iron conditions.

    PubMed

    Hopff, David; Wienkoop, Stefanie; Lüthje, Sabine

    2013-10-08

    Iron (Fe) homeostasis is essential for life and has been intensively investigated for dicots, while our knowledge for species in the Poaceae is fragmentary. This study presents the first proteome analysis (LC-MS/MS) of plasma membranes isolated from roots of 18-day old maize (Zea mays L.). Plants were grown under low and high Fe conditions in hydroponic culture. In total, 227 proteins were identified in control plants, whereas 204 proteins were identified in Fe deficient plants and 251 proteins in plants grown under high Fe conditions. Proteins were sorted by functional classes, and most of the identified proteins were classified as signaling proteins. A significant number of PM-bound redox proteins could be identified including quinone reductases, heme and copper-containing proteins. Most of these components were constitutive, and others could hint at an involvement of redox signaling and redox homeostasis by change in abundance. Energy metabolism and translation seem to be crucial in Fe homeostasis. The response to Fe deficiency includes proteins involved in development, whereas membrane remodeling and assembly and/or repair of Fe-S clusters is discussed for Fe toxicity. The general stress response appears to involve proteins related to oxidative stress, growth regulation, an increased rigidity and synthesis of cell walls and adaption of nutrient uptake and/or translocation. This article is part of a Special Issue entitled: Plant Proteomics in Europe.

  19. Aqueous polymer two-phase systems: effective tools for plasma membrane proteomics.

    PubMed

    Schindler, Jens; Nothwang, Hans Gerd

    2006-10-01

    Plasma membranes (PMs) are of particular importance for all living cells. They form a selectively permeable barrier to the environment. Many essential tasks of PMs are carried out by their proteinaceous components, including molecular transport, cell-cell interactions, and signal transduction. Due to the key role of these proteins for cellular function, they take center-stage in basic and applied research. A major problem towards in-depth identification and characterization of PM proteins by modern proteomic approaches is their low abundance and immense heterogeneity in different cells. Highly selective and efficient purification protocols are hence essential to any PM proteome analysis. An effective tool for preparative isolation of PMs is partitioning in aqueous polymer two-phase systems. In two-phase systems, membranes are separated according to differences in surface properties rather than size and density. Despite their rare application to the fractionation of animal tissues and cells, they represent an attractive alternative to conventional fractionation protocols. Here, we review the principles of partitioning using aqueous polymer two-phase systems and compare aqueous polymer two-phase systems with other methods currently used for the isolation of PMs.

  20. Proteomic analysis identifies interleukin 11 regulated plasma membrane proteins in human endometrial epithelial cells in vitro

    PubMed Central

    2011-01-01

    Background During the peri-implantation period, the embryo adheres to an adequately prepared or receptive endometrial surface epithelium. Abnormal embryo adhesion to the endometrium results in embryo implantation failure and infertility. Endometrial epithelial cell plasma membrane proteins critical in regulating adhesion may potentially be infertility biomarkers or targets for treating infertility. Interleukin (IL) 11 regulates human endometrial epithelial cells (hEEC) adhesion. Its production is abnormal in women with infertility. The objective of the study was to identify IL11 regulated plasma membrane proteins in hEEC in vitro using a proteomic approach. Methods Using a 2D-differential in-gel electrophoresis (DIGE) electrophoresis combined with LCMS/MS mass spectrometry approach, we identified 20 unique plasma membrane proteins differentially regulated by IL11 in ECC-1 cells, a hEEC derived cell line. Two IL11 regulated proteins with known roles in cell adhesion, annexin A2 (ANXA2) and flotillin-1 (FLOT1), were validated by Western blot and immunocytochemistry in hEEC lines (ECC-1 and an additional cell line, Ishikawa) and primary hEEC. Flotilin-1 was further validated by immunohistochemistry in human endometrium throughout the menstrual cycle (n = 6-8/cycle). Results 2D-DIGE analysis identified 4 spots that were significantly different between control and IL11 treated group. Of these 4 spots, there were 20 proteins that were identified with LCMS/MS. Two proteins; ANXA2 and FLOT1 were chosen for further analyses and have found to be significantly up-regulated following IL11 treatment. Western blot analysis showed a 2-fold and a 2.5-fold increase of ANXA2 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. Similarly, a 1.8-fold and a 2.3/2.4-fold increase was also observed for FLOT1 in hEEC membrane fraction of ECC-1 and Ishikawa cells respectively. In vitro, IL11 induced stronger ANXA2 expression on cell surface of primary hEEC and ECC-1 whilst

  1. Identification of potential biomarkers of hepatotoxicity by plasma proteome analysis of arsenic-exposed carp Labeo rohita.

    PubMed

    Banerjee, Sudeshna; Mahanty, Arabinda; Mohanty, Sasmita; Mazumder, Debendranath Guha; Cash, Phillip; Mohanty, Bimal Prasanna

    2017-08-15

    Arsenic (As) is a toxic environmental contaminant and potential human carcinogen. Chronic intake of arsenic-contaminated water and food leads to arsenicosis, a major public health problem in many parts of the world. Early detection of arsenic toxicity would greatly benefit patients; however, the detection of arsenicosis needs to be done early before onset of severe symptoms in which case the tools used for detection have to be both sensitive and reliable. In this context, the present study investigated plasma proteome changes in arsenic-exposed Labeo rohita, with the aim of identifying biomarkers for arsenicosis. Changes in the plasma proteome were investigated using gel-based proteomics technology. Using quantitative image analysis of the 2D proteome profiles, 14 unique spots were identified by MALDI-TOF/TOF MS and/or LC-MS/MS which included Apolipoprotein-A1 (Apo-A1) (6 spots), α-2 macroglobulin-like protein (A2ML) (2 spots), transferrin (TF) (3 spots) and warm-temperature acclimation related 65kDa protein (Wap65). The proteome data are available via ProteomeXchange with identifier PXD003404. Highly abundant protein spots identified in plasma from arsenic-exposed fish i.e. Apo-A1 (>10-fold), A2ML (7-fold) and Wap65 (>2-fold) indicate liver damage. It is proposed that a combination of these proteins could serve as useful biomarkers of hepatotoxicity and chronic liver disease due to arsenic exposure. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Evaluation of Multi-Protein Immunoaffinity Subtraction for Plasma Proteomics and Candidate Biomarker Discovery Using Mass Spectrometry

    PubMed Central

    Liu, Tao; Qian, Wei-Jun; Mottaz, Heather M.; Gritsenko, Marina A.; Norbeck, Angela D.; Moore, Ronald J.; Purvine, Samuel O.; Camp, David G.; Smith, Richard D.

    2007-01-01

    SUMMARY Strategies for removal of high-abundance proteins have been increasingly utilized in proteomic studies of serum/plasma and other body fluids to enhance the detection of low-abundance proteins and achieve broader proteome coverage; however, both the reproducibility and specificity of the high-abundance protein depletion process still represent common concerns. Here, we report a detailed evaluation of immunoaffinity subtraction performed applying the ProteomeLab IgY-12 system which is commonly used in human serum/plasma proteome characterization in combination with high resolution LC-MS/MS. Plasma samples were repeatedly processed implementing this system, and the resulting flow-through fractions and bound fractions were individually analyzed for comparison. The removal of target proteins by the immunoaffinity subtraction system and the overall process was highly reproducible. Non-target proteins, including one spiked protein standard (rabbit glyceraldehyde-3-phosphate dehydrogenase), were also observed to bind to the column at different levels, but in a reproducible manner. The results suggest that multi-protein immunoaffinity subtraction systems can be readily integrated into quantitative strategies to enhance detection of low-abundance proteins in biomarker discovery studies. PMID:16854842

  3. The proteome signature of the inflammatory breast cancer plasma membrane identifies novel molecular markers of disease

    PubMed Central

    Suárez-Arroyo, Ivette J; Feliz-Mosquea, Yismeilin R; Pérez-Laspiur, Juliana; Arju, Rezina; Giashuddin, Shah; Maldonado-Martínez, Gerónimo; Cubano, Luis A; Schneider, Robert J; Martínez-Montemayor, Michelle M

    2016-01-01

    Inflammatory Breast Cancer (IBC) is the most lethal form of breast cancer with a 35% 5-year survival rate. The accurate and early diagnosis of IBC and the development of targeted therapy against this deadly disease remain a great medical challenge. Plasma membrane proteins (PMPs) such as E-cadherin and EGFR, play an important role in the progression of IBC. Because the critical role of PMPs in the oncogenic processes they are the perfect candidates as molecular markers and targets for cancer therapies. In the present study, Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) followed by mass spectrometry analysis was used to compare the relative expression levels of membrane proteins (MP) between non-cancerous mammary epithelial and IBC cells, MCF-10A and SUM-149, respectively. Six of the identified PMPs were validated by immunoblotting using the membrane fractions of non-IBC and IBC cell lines, compared with MCF-10A cells. Immunohistochemical analysis using IBC, invasive ductal carcinoma or normal mammary tissue samples was carried out to complete the validation method in nine of the PMPs. We identified and quantified 278 MPs, 76% of which classified as PMPs with 1.3-fold or higher change. We identified for the first time the overexpression of the novel plasminogen receptor, PLGRKT in IBC and of the carrier protein, SCAMP3. Furthermore, we describe the positive relationship between L1CAM expression and metastasis in IBC patients and the role of SCAMP3 as a tumor-related protein. Overall, the membrane proteomic signature of IBC reflects a global change in cellular organization and suggests additional strategies for cancer progression. Together, this study provides insight into the specialized IBC plasma membrane proteome with the potential to identify a number of novel therapeutic targets for IBC. PMID:27648361

  4. Evaluation of Multi-Protein Immunoaffinity Subtraction for Plasma Proteomics and Candidate Biomarker Discovery Using Mass Spectrometry

    SciTech Connect

    Liu, Tao; Qian, Weijun; Mottaz, Heather M.; Gritsenko, Marina A.; Norbeck, Angela D.; Moore, Ronald J.; Purvine, Samuel O.; Camp, David G.; Smith, Richard D.

    2006-11-01

    The detection of low-abundance protein disease biomarkers from human blood poses significant challenges due to the high dynamic range of protein concentrations that span more than 10 orders of magnitude, as well as the extreme complexity of the serum/plasma proteome. Therefore, experimental strategies that include the removal of high-abundance proteins have been increasingly utilized in proteomic studies of serum, plasma, and other body fluids to enhance detection of low-abundance proteins and achieve broader proteome coverage. However, both the specificity and reproducibility of the high-abundance protein depletion process represent common concerns. Here, we report a detailed evaluation of the performance of two commercially available immunoaffinity subtraction systems commonly used in human serum/plasma proteome characterization by high resolution LC-MS/MS. One system uses mammalian IgG antibodies to remove six of the most abundant plasma proteins, and the other uses chicken immunoglobulin yolk (IgY) antibodies to remove twelve of the most abundant plasma proteins. Plasma samples were repeatedly processed using these two systems, and the resulting flow-through fractions and bound fractions were individually analyzed for comparison. Removal of target proteins by both immunoaffinity subtraction systems proved reproducible and efficient. Nontarget proteins, including spiked protein standards, were also observed to bind to the columns, but in a fairly reproducible manner. The results suggest that these multi-protein immunoaffinity subtraction systems are both highly effective and reproducible for removing high-abundance proteins and therefore, can be readily integrated into quantitative strategies to enhance detection of low-abundance proteins in biomarker discovery studies.

  5. Comparative Proteome Analyses of Human Plasma Following in vivo Lipopolysaccharide Administration Using Multidimensional Separations Coupled with Tandem Mass Spectrometry

    PubMed Central

    Qian, Wei-Jun; Jacobs, Jon M.; Camp, David G.; Monroe, Matthew E.; Moore, Ronald J.; Gritsenko, Marina A.; Calvano, Steve E.; Lowry, Stephen F.; Xiao, Wenzhong; Moldawer, Lyle L.; Davis, Ronald W.; Tompkins, Ronald G.; Smith, Richard D.

    2007-01-01

    There is significant interest in characterization of the human plasma proteome due to its potential for providing biomarkers applicable to clinical diagnosis and treatment and for gaining a better understanding of human diseases. We describe here a strategy for the comparative proteome analyses of human plasma, which is applicable to biomarker identifications for various disease states. Multidimensional liquid chromatography-mass spectrometry has been applied to make comparative proteome analyses of plasma samples from an individual prior to and 9 h after lipopolysaccharide (LPS) administration. Peptide peak areas and the number of peptide identifications for each protein were used to evaluate the reproducibility of LC-MS/MS and to compare relative changes in protein concentration between the samples following LPS treatment. A total of 804 distinct plasma proteins (not including immunoglobulins) were confidently identified with 32 proteins observed to be significantly increased in concentration following LPS administration, including several known inflammatory response or acute-phase mediators such as C-reactive protein, serum amyloid A and A2, LPS-binding protein, LPS-responsive and beige-like anchor protein, hepatocyte growth factor activator, and von Willebrand factor, and thus constituting potential biomarkers for inflammatory response. PMID:15627965

  6. PROTEOMICS ANALYSIS OF OVEREXPRESSED PLASMA PROTEINS IN RESPONSE TO COLD ACCLIMATION IN Ostrinia furnacalis.

    PubMed

    Shang, Qingli; Pan, Yiou; Peng, Tianfei; Yang, Shuang; Lu, Xin; Wang, Zhenying; Xi, Jinghui

    2015-12-01

    Many insects in temperate regions overwinter in diapause. In these insects, one of the metabolic adaptations to cold stress is the synthesis of responsive proteins. Using proteomic analysis, an investigation aimed to a better understanding of the molecular adaptation mechanisms to cold stress was carried out in Ostrinia furnacalis larva. Proteins were extracted from the larval hemolymph collected from both control and overwintering larva. By polyethylene glycol precipitation, approximately 560 protein spots were separated and visualized on two-dimensional (2D) gels after silver staining. Eighteen protein spots were found to be upregulated in overwinter larval plasma in different patterns. As an initial work, 13 of these proteins were identified using MALDI TOF/TOF MS. The differentially overexpressed proteins include heat shock 70 kDa cognate protein, small heat shock protein (sHSP), putative aliphatic nitrilase, arginine kinase, phosphoglyceromutase, triosephosphateisomerase, and glutathione transferase. Alterations in the levels of these proteins were further confirmed by qPCR. This study is the first analysis of differentially expressed plasma proteins in O. furnacalis diapause larvae under extremely low temperature conditions and gives new insights into the acclimation mechanisms responsive to cold stress. Our results also support the idea that energy metabolism, alanine and proline metabolism, and antioxidative reaction act in the cold acclimation of O. furnacalis diapause larvae. © 2015 Wiley Periodicals, Inc.

  7. Proteomic Changes in the Plasma of Broiler Chickens with Femoral Head Necrosis

    PubMed Central

    Packialakshmi, Balamurugan; Liyanage, Rohana; Lay, Jackson O.; Okimoto, Ronald; Rath, Narayan C.

    2016-01-01

    Femoral head necrosis (FHN) is a skeletal problem in broiler chickens, where the proximal femoral head cartilage shows susceptibility to separation from its growth plate. The selected birds with FHN showed higher body weights and reduced plasma cholesterol. The proteomic differences in the plasma of healthy and FHN-affected chickens were explored using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography/electrospray ionization-tandem mass spectrometry (LC-MS/MS) to prospect for protein biomarkers. We isolated two differentially expressed low molecular weight proteins and identified them by MALDI peptide mass fingerprinting as fibrinogen- and fetuin-derived peptides, respectively. These peptides were reduced in birds susceptible to femoral head problems. Quantitation of LC-MS/MS spectra showed elevated levels of gallinacin-9, apolipoprotein A1, and hemoglobin and reduced levels of alpha-1-acid glycoprotein, albumin, and SPINK7 proteins in FHN. These results suggest that the bodyweight and the lipid profiles along with the above proteins can be useful as noninvasive biomarkers of FHN. PMID:27147818

  8. Proteome changes in the plasma of Pieris rapae parasitized by the endoparasitoid wasp Pteromalus puparum *

    PubMed Central

    Zhu, Jia-ying; Fang, Qi; Ye, Gong-yin; Hu, Cui

    2011-01-01

    Parasitism by the endoparasitoid wasp Pteromalus puparum causes alterations in the plasma proteins of Pieris rapae. Analysis of plasma proteins using a proteomic approach showed that seven proteins were differentially expressed in the host pupae after 24-h parasitism. They were masquerade-like serine proteinase homolog (MSPH), enolase (Eno), bilin-binding protein (BBP), imaginal disc growth factor (IDGF), ornithine decarboxylase (ODC), cellular retinoic acid binding protein (CRABP), and one unknown function protein. The full length cDNA sequences of MSPH, Eno, and BBP were successfully cloned using rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicated that the transcript levels of MSPH and BBP in the fat bodies of host pupae were inducible in response to the parasitism and their variations were consistent with translational changes of these genes after parasitism, while the transcript levels of Eno and IDGF were not affected by parasitism. This study will contribute to the better understanding of the molecular bases of parasitoid-induced host alterations associated with innate immune responses, detoxification, and energy metabolism. PMID:21265041

  9. Plasma protein changes in horse after prolonged physical exercise: a proteomic study.

    PubMed

    Scoppetta, Fausto; Tartaglia, Micaela; Renzone, Giovanni; Avellini, Luca; Gaiti, Alberto; Scaloni, Andrea; Chiaradia, Elisabetta

    2012-07-19

    Physical exercise induces various stress responses and metabolic adaptations that have not yet been completely elucidated. Novel biomarkers are needed in sport veterinary medicine to monitor training levels and to detect subclinical conditions that can develop into exercise-related diseases. In this study, protein modifications in horse plasma induced by prolonged, aerobic physical exercise were investigated by using a proteomic approach based on 2-DE and combined mass spectrometry procedures. Thirty-eight protein spots, associated with expression products of 13 genes, showed significant quantitative changes; spots identified as membrane Cu amine oxidase, α-1 antitrypsin, α-1 antitrypsin-related protein, caeruloplasmin, α-2 macroglobulin and complement factor C4 were augmented in relative abundance after the race, while haptoglobin β chain, apolipoprotein A-I, transthyretin, retinol binding protein 4, fibrinogen γ chain, complement factor B and albumin fragments were reduced. These results indicate that prolonged physical exercise affects plasma proteins involved in pathways related to inflammation, coagulation, immune modulation, oxidant/antioxidant activity and cellular and vascular damage, with consequent effects on whole horse metabolism.

  10. The plasma membrane proteome of Medicago truncatula roots as modified by arbuscular mycorrhizal symbiosis.

    PubMed

    Aloui, Achref; Recorbet, Ghislaine; Lemaître-Guillier, Christelle; Mounier, Arnaud; Balliau, Thierry; Zivy, Michel; Wipf, Daniel; Dumas-Gaudot, Eliane

    2017-07-19

    In arbuscular mycorrhizal (AM) roots, the plasma membrane (PM) of the host plant is involved in all developmental stages of the symbiotic interaction, from initial recognition to intracellular accommodation of intra-radical hyphae and arbuscules. Although the role of the PM as the agent for cellular morphogenesis and nutrient exchange is especially accentuated in endosymbiosis, very little is known regarding the PM protein composition of mycorrhizal roots. To obtain a global overview at the proteome level of the host PM proteins as modified by symbiosis, we performed a comparative protein profiling of PM fractions from Medicago truncatula roots either inoculated or not with the AM fungus Rhizophagus irregularis. PM proteins were isolated from root microsomes using an optimized discontinuous sucrose gradient; their subsequent analysis by liquid chromatography followed by mass spectrometry (MS) identified 674 proteins. Cross-species sequence homology searches combined with MS-based quantification clearly confirmed enrichment in PM-associated proteins and depletion of major microsomal contaminants. Changes in protein amounts between the PM proteomes of mycorrhizal and non-mycorrhizal roots were monitored further by spectral counting. This workflow identified a set of 82 mycorrhiza-responsive proteins that provided insights into the plant PM response to mycorrhizal symbiosis. Among them, the association of one third of the mycorrhiza-responsive proteins with detergent-resistant membranes pointed at partitioning to PM microdomains. The PM-associated proteins responsive to mycorrhization also supported host plant control of sugar uptake to limit fungal colonization, and lipid turnover events in the PM fraction of symbiotic roots. Because of the depletion upon symbiosis of proteins mediating the replacement of phospholipids by phosphorus-free lipids in the plasmalemma, we propose a role of phosphate nutrition in the PM composition of mycorrhizal roots.

  11. Identification of putative fertility markers in seminal plasma of crossbred bulls through differential proteomics.

    PubMed

    Aslam, M K Muhammad; Kumaresan, A; Sharma, Vinay K; Tajmul, Md; Chhillar, Shivani; Chakravarty, A K; Manimaran, A; Mohanty, T K; Srinivasan, A; Yadav, Savita

    2014-12-01

    Sub-fertility is a major problem in crossbred bulls leading to disintegration of breeding systems and huge economic loss. Identification of some potential biomarkers to determine the latent fertility of bulls accurately has long been the interest of researchers. In this study, we analyzed the proteome of seminal plasma (SP) from bulls with varying fertility to identify the fertility-associated proteins. The proteomic profile of high- and low-fertile bulls was compared by two-dimensional difference gel electrophoresis and differentially expressed proteins were identified through matrix-assisted laser desorption/ionization-time of flight/mass spectrometry. Out of the 18 differentially expressed proteins (P < 0.05), 9 were overexpressed in SP of high-fertile bulls and 9 were overexpressed in SP of low-fertile bulls. The differential expressions ranged from 1.5- to 5.5-fold between the two groups, where protection of telomeres-1 protein (POT1) was highly overexpressed (2.9-fold) in high-fertile group and prostaglandin E2 receptor EP3 (PTGER3) was highly abundant (5.5-fold) in low-fertile group. The protein interaction network was elucidated using STRING software tool, and the functional bioinformatics analysis was done using Blast2Go software. Most of the differentially expressed proteins were found to be involved in cellular processes and biological regulation with binding and catalytic function. It is inferred that the expression of certain proteins in the SP varied with bull fertility, and concurrent appraisal of their expression along with other fertility assays may help in determining bull fertility. Copyright © 2014 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2016-09-02

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

  13. A partial proteome reference map of the wine lactic acid bacterium Oenococcus oeni ATCC BAA-1163.

    PubMed

    Mohedano, María de la Luz; Russo, Pasquale; de Los Ríos, Vivian; Capozzi, Vittorio; Fernández de Palencia, Pilar; Spano, Giuseppe; López, Paloma

    2014-02-26

    Oenococcus oeni is the main lactic acid bacterium that carries out the malolactic fermentation in virtually all red wines and in some white and sparkling wines. Oenococcus oeni possesses an array of metabolic activities that can modify the taste and aromatic properties of wine. There is, therefore, industrial interest in the proteins involved in these metabolic pathways and related transport systems of this bacterium. In this work, we report the characterization of the O. oeni ATCC BAA-1163 proteome. Total and membrane protein preparations from O. oeni were standardized and analysed by two-dimensional gel electrophoresis. Using tandem mass spectrometry, we identified 224 different spots corresponding to 152 unique proteins, which have been classified by their putative function and subjected to bioinformatics analysis.

  14. A reference proteomic database of Lactobacillus plantarum CMCC-P0002.

    PubMed

    Zhu, Li; Hu, Wei; Liu, Datao; Tian, Wanhong; Yu, Gang; Liu, Xiankai; Wang, Jie; Feng, Erling; Zhang, Xuemin; Chen, Bei; Zeng, Ming; Wang, Hengliang

    2011-01-01

    Lactobacillus plantarum is a widespread probiotic bacteria found in many fermented food products. In this study, the whole-cell proteins and secretory proteins of L. plantarum were separated by two-dimensional electrophoresis method. A total of 434 proteins were identified by tandem mass spectrometry, including a plasmid-encoded hypothetical protein pLP9000_05. The information of first 20 highest abundance proteins was listed for the further genetic manipulation of L. plantarum, such as construction of high-level expressions system. Furthermore, the first interaction map of L. plantarum was established by Blue-Native/SDS-PAGE technique. A heterodimeric complex composed of maltose phosphorylase Map3 and Map2, and two homodimeric complexes composed of Map3 and Map2 respectively, were identified at the same time, indicating the important roles of these proteins. These findings provided valuable information for the further proteomic researches of L. plantarum.

  15. The plasma membrane-enriched fraction proteome response during adaptation to hydrogen peroxide in Saccharomyces cerevisiae.

    PubMed

    Pedroso, Nuno; Gomes-Alves, Patrícia; Marinho, H Susana; Brito, Verônica B; Boada, Cristina; Antunes, Fernando; Herrero, Enrique; Penque, Deborah; Cyrne, Luísa

    2012-10-01

    In Saccharomyces cerevisiae, adaptation to hydrogen peroxide (H₂O₂) decreases plasma membrane permeability to H₂O₂, changes its lipid composition and reorganizes ergosterol-rich microdomains by a still unknown mechanism. Here we show, by a quantitative analysis of the H₂O₂-induced adaptation effect on the S. cerevisiae plasma membrane-enriched fraction proteome, using two-dimensional gel electrophoresis, that 44 proteins are differentially expressed. Most of these proteins were regulated at a post-transcriptional level. Fourteen of these proteins contain redox-sensitive cysteine residues and nine proteins are associated with lipid and vesicle traffic. In particular, three proteins found in eisosomes and in the eisosome-associated membrane compartment occupied by Can1p were up-regulated (Pil1p, Rfs1p and Pst2p) during adaptation to H₂O₂. Survival studies after exposure to lethal H₂O₂ doses using yeast strains bearing a gene deletion corresponding to proteins associated to lipid and vesicle traffic demonstrated for the first time that down-regulation of Kes1p, Vps4p and Ynl010wp and up-regulation of Atp1 and Atp2 increases resistance to H₂O₂. Moreover, for the pil1Δ strain, H₂O₂ at low levels produces a hormetic effect by increasing proliferation. In conclusion, these data further confirms the plasma membrane as an active cellular site during adaptation to H₂O₂ and shows that proteins involved in lipid and vesicle traffic are important mediators of H₂O₂ adaptation.

  16. Selecting Optimal Peptides for Targeted Proteomic Experiments in Human Plasma Using in vitro Synthesized Proteins as Analytical Standards

    PubMed Central

    Bollinger, James G.; Stergachis, Andrew B.; Johnson, Richard S.; Egertson, Jarrett D.; MacCoss, Michael J.

    2017-01-01

    Summary In targeted proteomics, the development of robust methodologies is dependent upon the selection of a set of optimal peptides for each protein-of-interest. Unfortunately, predicting which peptides and respective product ion transitions provide the greatest signal-to-noise ratio in a particular assay matrix is complicated. Using in vitro synthesized proteins as analytical standards, we report here an empirically driven method for the selection of said peptides in a human plasma assay matrix. PMID:26867746

  17. Selecting Optimal Peptides for Targeted Proteomic Experiments in Human Plasma Using In Vitro Synthesized Proteins as Analytical Standards.

    PubMed

    Bollinger, James G; Stergachis, Andrew B; Johnson, Richard S; Egertson, Jarrett D; MacCoss, Michael J

    2016-01-01

    In targeted proteomics, the development of robust methodologies is dependent upon the selection of a set of optimal peptides for each protein-of-interest. Unfortunately, predicting which peptides and respective product ion transitions provide the greatest signal-to-noise ratio in a particular assay matrix is complicated. Using in vitro synthesized proteins as analytical standards, we report here an empirically driven method for the selection of said peptides in a human plasma assay matrix.

  18. Towards a membrane proteome in Drosophila: a method for the isolation of plasma membrane

    PubMed Central

    2010-01-01

    Background The plasma membrane (PM) is a compartment of significant interest because cell surface proteins influence the way in which a cell interacts with its neighbours and its extracellular environment. However, PM is hard to isolate because of its low abundance. Aqueous two-phase affinity purification (2PAP), based on PEG/Dextran two-phase fractionation and lectin affinity for PM-derived microsomes, is an emerging method for the isolation of high purity plasma membranes from several vertebrate sources. In contrast, PM isolation techniques in important invertebrate genetic model systems, such as Drosophila melanogaster, have relied upon enrichment by density gradient centrifugation. To facilitate genetic investigation of activities contributing to the content of the PM sub-proteome, we sought to adapt 2PAP to this invertebrate model to provide a robust PM isolation technique for Drosophila. Results We show that 2PAP alone does not completely remove contaminating endoplasmic reticulum and mitochondrial membrane. However, a novel combination of density gradient centrifugation plus 2PAP results in a robust PM preparation. To demonstrate the utility of this technique we isolated PM from fly heads and successfully identified 432 proteins using MudPIT, of which 37% are integral membrane proteins from all compartments. Of the 432 proteins, 22% have been previously assigned to the PM compartment, and a further 34% are currently unassigned to any compartment and represent candidates for assignment to the PM. The remainder have previous assignments to other compartments. Conclusion A combination of density gradient centrifugation and 2PAP results in a robust, high purity PM preparation from Drosophila, something neither technique can achieve on its own. This novel preparation should lay the groundwork for the proteomic investigation of the PM in different genetic backgrounds in Drosophila. Our results also identify two key steps in this procedure: The optimization of

  19. The effects of eating marine- or vegetable-fed farmed trout on the human plasma proteome profiles of healthy men.

    PubMed

    Rentsch, Maria L; Lametsch, René; Bügel, Susanne; Jessen, Flemming; Lauritzen, Lotte

    2015-02-28

    Most human intervention studies have examined the effects on a subset of risk factors, some of which may require long-term exposure. The plasma proteome may reflect the underlying changes in protein expression and activation, and this could be used to identify early risk markers. The aim of the present study was to evaluate the impact of regular fish intake on the plasma proteome. We recruited thirty healthy men aged 40 to 70 years, who were randomly allocated to a daily meal of chicken or trout raised on vegetable or marine feeds. Blood samples were collected before and after 8 weeks of intervention, and after the removal of the twelve most abundant proteins, plasma proteins were separated by two-dimensional gel electrophoresis. Protein spots < 66 kDa with a pI > 4·3 visualised by silver staining were matched by two-dimensional imaging software. Within-subject changes in spots were compared between the treatment groups. Differentially affected spots were identified by matrix-assisted laser desorption ionisation-time of flight/time of flight MS and the human Swiss-Prot database. We found 23/681 abundant plasma protein spots, which were up- or down-regulated by the dietary treatment (P < 0·05, q < 0·30), and eighteen of these were identified. In each trout group, ten spots differed from those in subjects given the chicken meal, but only three of these were common, and only one spot differed between the two trout groups. In both groups, the affected plasma proteins were involved in biological processes such as regulation of vitamin A and haem transport, blood fibrinolysis and oxidative defence. Thus, regular fish intake affects the plasma proteome, and the changes may indicate novel mechanisms of effect.

  20. Proteomic analysis of the soluble fraction from human corneal fibroblasts with reference to ocular transparency.

    PubMed

    Karring, Henrik; Thøgersen, Ida B; Klintworth, Gordon K; Enghild, Jan J; Møller-Pedersen, Torben

    2004-07-01

    The transparent corneal stroma contains a population of corneal fibroblasts termed keratocytes, which are interspersed between the collagen lamellae. Under normal conditions, the keratocytes are quiescent and transparent. However, after corneal injury the keratocytes become activated and transform into backscattering wound-healing fibroblasts resulting in corneal opacification. At present, the most popular hypothesis suggests that particular abundant water-soluble proteins called enzyme-crystallins are involved in maintaining corneal cellular transparency. Specifically, corneal haze development is thought to be related to low levels of cytoplasmic enzyme-crystallins in reflective corneal fibroblasts. To further investigate this hypothesis, we have used a proteomic approach to identify the most abundant water-soluble proteins in serum-cultured human corneal fibroblasts that represent an in vitro model of the reflective wound-healing keratocyte phenotype. Densitometry of one-dimensional gels revealed that no single protein isoform exceeded 5% of the total water-soluble protein fraction, which is the qualifying property of a corneal enzyme-crystallin according to the current definition. This result indicates that wound-healing corneal fibroblasts do not contain enzyme-crystallins. A total of 254 protein identifications from two-dimensional gels were performed representing 118 distinct proteins. Proteins protecting against oxidative stress and protein misfolding were prominent, suggesting that these processes may participate in the generation of cytoplasmic light-scattering from corneal fibroblasts.

  1. Long-term heat stress induces the inflammatory response in dairy cows revealed by plasma proteome analysis.

    PubMed

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

    2016-03-04

    In this work we employed a comparative proteomic approach to evaluate seasonal heat stress and investigate proteomic alterations in plasma of dairy cows. Twelve lactating Holstein dairy cows were used and the treatments were: heat stress (n = 6) in hot summer (at the beginning of the moderate heat stress) and no heat stress (n = 6) in spring natural ambient environment, respectively. Subsequently, heat stress treatment lasted 23 days (at the end of the moderate heat stress) to investigate the alterations of plasma proteins, which might be employed as long-term moderate heat stress response in dairy cows. Changes in plasma proteins were analyzed by two-dimensional electrophoresis (2-DE) combined with mass spectrometry. Analysis of the properties of the identified proteins revealed that the alterations of plasma proteins were related to inflammation in long-term moderate heat stress. Furthermore, the increase in plasma tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) directly demonstrated that long-term moderate heat stress caused an inflammatory response in dairy cows.

  2. Biological variation and reference change values of feline plasma biochemistry analytes.

    PubMed

    Baral, Randolph M; Dhand, Navneet K; Freeman, Kathleen P; Krockenberger, Mark B; Govendir, Merran

    2014-04-01

    This is the first report concerning biological variation and reference change values of feline plasma biochemistry components in the peer-reviewed literature. Biological variation refers to inherent physiological variation of analytes. The ratio of individual biological variation to group biological variation is referred to as an analyte's index of individuality. This index determines the suitability of an analyte to be assessed in relation to population- or subject-based reference intervals. A subject-based reference interval is referred to as a reference change value or critical difference, and is calculated from individual biological variation. Fourteen cats were sampled for plasma biochemistry analysis once weekly for 6 weeks. Samples were stored and then tested at the same time. Results were assessed in duplicate and coefficients of variation for each analyte were isolated to distinguish variation within each subject, between all subjects and by the analyser. From these results, an index of individuality and reference change values were determined for each analyte. Five plasma biochemistry analytes (alkaline phosphatase, alanine aminotransferase, cholesterol, creatinine and globulin) had high individuality and, therefore, subject-based reference intervals are more appropriate; only one analyte (sodium) had low individuality, indicating that population-based reference intervals are appropriate. Most analytes had intermediate individuality so population-based reference intervals should be assessed in relation to subject-based reference intervals. The results of this study demonstrate high individuality for most analytes and, therefore, that population-based reference intervals are of limited utility for most biochemical analytes in cats.

  3. 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. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Applications in Quantitative Proteomics.

    PubMed

    Chahrour, Osama; Malone, John

    2017-01-01

    Recent advances in inductively coupled plasma mass spectrometry (ICP-MS) hyphenated to different separation techniques have promoted it as a valuable tool in protein/peptide quantification. These emerging ICP-MS applications allow absolute quantification by measuring specific elemental responses. One approach quantifies elements already present in the structure of the target peptide (e.g. phosphorus and sulphur) as natural tags. Quantification of these natural tags allows the elucidation of the degree of protein phosphorylation in addition to absolute protein quantification. A separate approach is based on utilising bi-functional labelling substances (those containing ICP-MS detectable elements), that form a covalent chemical bond with the protein thus creating analogs which are detectable by ICP-MS. Based on the previously established stoichiometries of the labelling reagents, quantification can be achieved. This technique is very useful for the design of precise multiplexed quantitation schemes to address the challenges of biomarker screening and discovery. This review discusses the capabilities and different strategies to implement ICP-MS in the field of quantitative proteomics. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  5. Effects of ω-3 Polyunsaturated Fatty Acids on Plasma Proteome in Rett Syndrome

    PubMed Central

    De Felice, Claudio; Cortelazzo, Alessio; Guerranti, Roberto; Pecorelli, Alessandra; Durand, Thierry; Galano, Jean-Marie; Oger, Camille; Montomoli, Barbara; Landi, Claudia; Valacchi, Giuseppe; Ciccoli, Lucia; Hayek, Joussef

    2013-01-01

    The mechanism of action of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) is only partially known. Prior reports suggest a partial rescue of clinical symptoms and oxidative stress (OS) alterations following ω-3 PUFAs supplementation in patients with Rett syndrome (RTT), a devastating neurodevelopmental disorder with transient autistic features, affecting almost exclusively females and mainly caused by sporadic mutations in the gene encoding the methyl CpG binding protein 2 (MeCP2) protein. Here, we tested the hypothesis that ω-3 PUFAs may modify the plasma proteome profile in typical RTT patients with MECP2 mutations and classic phenotype. A total of 24 RTT girls at different clinical stages were supplemented with ω-3 PUFAs as fish oil for 12 months and compared to matched healthy controls. The expression of 16 proteins, mainly related to acute phase response (APR), was changed at the baseline in the untreated patients. Following ω-3 PUFAs supplementation, the detected APR was partially rescued, with the expression of 10 out of 16 (62%) proteins being normalized. ω-3 PUFAs have a major impact on the modulation of the APR in RTT, thus providing new insights into the role of inflammation in autistic disorders and paving the way for novel therapeutic strategies. PMID:24385686

  6. Use of proteomics for validation of the isolation process of clotting factor IX from human plasma.

    PubMed

    Clifton, James; Huang, Feilei; Gaso-Sokac, Dajana; Brilliant, Kate; Hixson, Douglas; Josic, Djuro

    2010-01-03

    The use of proteomic techniques in the monitoring of different production steps of plasma-derived clotting factor IX (pd F IX) was demonstrated. The first step, solid-phase extraction with a weak anion-exchange resin, fractionates the bulk of human serum albumin (HSA), immunoglobulin G, and other non-binding proteins from F IX. The proteins that strongly bind to the anion-exchange resin are eluted by higher salt concentrations. In the second step, anion-exchange chromatography, residual HSA, some proteases and other contaminating proteins are separated. In the last chromatographic step, affinity chromatography with immobilized heparin, the majority of the residual impurities are removed. However, some contaminating proteins still remain in the eluate from the affinity column. The next step in the production process, virus filtration, is also an efficient step for the removal of residual impurities, mainly high molecular weight proteins, such as vitronectin and inter-alpha inhibitor proteins. In each production step, the active component, pd F IX and contaminating proteins are monitored by biochemical and immunochemical methods and by LC-MS/MS and their removal documented. Our methodology is very helpful for further process optimization, rapid identification of target proteins with relatively low abundance, and for the design of subsequent steps for their removal or purification.

  7. Grape berry plasma membrane proteome analysis and its differential expression during ripening.

    PubMed

    Zhang, Jiangwei; Ma, Huiqin; Feng, Jidong; Zeng, Lei; Wang, Zeng; Chen, Shangwu

    2008-01-01

    High purity berry plasma membranes (PMs) of Vitis vinifera L. cv. Cabernet Sauvignon were isolated by two-phase partitioning of microsome fractions at different stages of berry ripening. PM proteins resolvable by the detergent cocktail of CHAPS and ASB-14 were separated by two-dimensional electrophoresis. A total of 119 protein spots from pre-véraison berry PMs on 2-D gels detected with silver staining were subjected to MALDI-TOF mass spectrometry analysis. Sixty-two spots were identified as putative PM proteins, with 1-6 predicted transmembrane helices, including true PM proteins such as ATP synthase, ABC transporters, and GTP-binding proteins reported in plants. They were then grouped into eight functional categories, mainly involved in transport, metabolism, signal transduction, and protein synthesis. Another 11 spots were identified as proteins of unknown function. The véraison and post-véraison samples stained 98 and 86 spots on the gels, respectively. During the berry ripening process, total PM protein content gradually decreased. Among all identified proteins, 12 showed significant differences in terms of their relative abundance. Increasing ubiquitin proteolysis and cytoskeleton proteins were observed from pre-véraison to post-véraison. Zeatin O-glucosyltransferase peaked at véraison, while ubiquitin-conjugating enzyme E2-21 was down-regulated at this stage. This proteome research provides the first information on PM protein characterization during the grape berry ripening process.

  8. Role of DHA in aging-related changes in mouse brain synaptic plasma membrane proteome.

    PubMed

    Sidhu, Vishaldeep K; Huang, Bill X; Desai, Abhishek; Kevala, Karl; Kim, Hee-Yong

    2016-05-01

    Aging has been related to diminished cognitive function, which could be a result of ineffective synaptic function. We have previously shown that synaptic plasma membrane proteins supporting synaptic integrity and neurotransmission were downregulated in docosahexaenoic acid (DHA)-deprived brains, suggesting an important role of DHA in synaptic function. In this study, we demonstrate aging-induced synaptic proteome changes and DHA-dependent mitigation of such changes using mass spectrometry-based protein quantitation combined with western blot or messenger RNA analysis. We found significant reduction of 15 synaptic plasma membrane proteins in aging brains including fodrin-α, synaptopodin, postsynaptic density protein 95, synaptic vesicle glycoprotein 2B, synaptosomal-associated protein 25, synaptosomal-associated protein-α, N-methyl-D-aspartate receptor subunit epsilon-2 precursor, AMPA2, AP2, VGluT1, munc18-1, dynamin-1, vesicle-associated membrane protein 2, rab3A, and EAAT1, most of which are involved in synaptic transmission. Notably, the first 9 proteins were further reduced when brain DHA was depleted by diet, indicating that DHA plays an important role in sustaining these synaptic proteins downregulated during aging. Reduction of 2 of these proteins was reversed by raising the brain DHA level by supplementing aged animals with an omega-3 fatty acid sufficient diet for 2 months. The recognition memory compromised in DHA-depleted animals was also improved. Our results suggest a potential role of DHA in alleviating aging-associated cognitive decline by offsetting the loss of neurotransmission-regulating synaptic proteins involved in synaptic function. Published by Elsevier Inc.

  9. Proteomic analysis of plasma membrane proteins in wheat roots exposed to phenanthrene.

    PubMed

    Shen, Yu; Du, Jiangxue; Yue, Le; Zhan, Xinhua

    2016-06-01

    Polycyclic aromatic hydrocarbons (PAHs) are potentially carcinogenic and toxic to humans through ingestion of contaminated food crops. PAHs can enter crop roots through proton/PAH symporters; however, to date, the symporter remains unclear. Here we reveal, for the first time, the plasma membrane proteome of Triticum aestivum seedling roots in response to phenanthrene (a model PAH) exposure. Two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF/TOF-MS and protein database search engines were employed to analyze and identify phenanthrene-responsive proteins. Over 192 protein spots are reproducibly detected in each gel, while 8 spots are differentially expressed under phenanthrene treatment. Phenanthrene induces five up-regulated proteins distinguished as 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase 2, enolase, heat shock protein 80-2, probable mediator of RNA polymerase II transcription subunit 37e (heat shock 70-kDa protein 1), and lactoylglutathione lyase. Three proteins identified as adenosine kinase 2, 4-hydroxy-7-methoxy-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl glucoside beta-D-glucosidase 1c, and glyceraldehyde-3-phosphate dehydrogenase 3 are down-regulated under exposure to phenanthrene. The up-regulated proteins are related to plant defense response, antioxidant system, and glycolysis. The down-regulated proteins involve the metabolism of high-energy compounds and plant growth. Magnesium, which is able to bind to enolase, can enhance the transport of phenanthrene into wheat roots. Therefore, it is concluded that phenanthrene can induce differential expression of proteins in relation to carbohydrate metabolism, self-defense, and plant growth on wheat root plasma membrane. This study not only provides novel insights into PAH uptake by plant roots and PAH stress responses, but is also a good starting point for further determination and analyses of their functions using genetic and other approaches.

  10. Targeted Proteomic Quantitation of the Absolute Expression and Turnover of Cystic Fibrosis Transmembrane Conductance Regulator in the Apical Plasma Membrane

    PubMed Central

    2015-01-01

    Deficient chloride transport through cystic fibrosis (CF) transmembrane conductance regulator (CFTR) causes lethal complications in CF patients. CF is the most common autosomal recessive genetic disease, which is caused by mutations in the CFTR gene; thus, CFTR mutants can serve as primary targets for drugs to modulate and rescue the ion channel’s function. The first step of drug modulation is to increase the expression of CFTR in the apical plasma membrane (PM); thus, accurate measurement of CFTR in the PM is desired. This work reports a tandem enrichment strategy to prepare PM CFTR and uses a stable isotope labeled CFTR sample as the quantitation reference to measure the absolute amount of apical PM expression of CFTR in CFBE 41o- cells. It was found that CFBE 41o- cells expressing wild-type CFTR (wtCFTR), when cultured on plates, had 2.9 ng of the protein in the apical PM per million cells; this represented 10% of the total CFTR found in the cells. When these cells were polarized on filters, the apical PM expression of CFTR increased to 14%. Turnover of CFTR in the apical PM of baby hamster kidney cells overexpressing wtCFTR (BHK-wtCFTR) was also quantified by targeted proteomics based on multiple reaction monitoring mass spectrometry; wtCFTR had a half-life of 29.0 ± 2.5 h in the apical PM. This represents the first direct measurement of CFTR turnover using stable isotopes. The absolute quantitation and turnover measurements of CFTR in the apical PM can significantly facilitate understanding the disease mechanism of CF and thus the development of new disease-modifying drugs. Absolute CFTR quantitation allows for direct result comparisons among analyses, analysts, and laboratories and will greatly amplify the overall outcome of CF research and therapy. PMID:25227318

  11. Differential effects of growth hormone versus insulin-like growth factor-I on the mouse plasma proteome.

    PubMed

    Ding, Juan; List, Edward O; Bower, Brian D; Kopchick, John J

    2011-10-01

    The GH/IGF-I axis has both pre- and postpubertal metabolic effects. However, the differential effects of GH and/or IGF-I on animal physiology or the plasma proteome are still being unraveled. In this report, we analyzed several physiological effects along with the plasma proteome after treatment of mice with recombinant bovine GH or recombinant human IGF-I. GH and IGF-I showed similar effects in increasing body length, body weight, lean and fluid masses, and organ weights including muscle, kidney, and spleen. However, GH significantly increased serum total cholesterol, whereas IGF-I had no effect on it. Both acute and longer-term effects on the plasma proteome were determined. Proteins found to be significantly changed by recombinant bovine GH and/or recombinant human IGF-I injections were identified by mass spectrometry (MS) and MS/MS. The identities of these proteins were further confirmed by Western blotting analysis. Isoforms of apolipoprotein A4, apolipoprotein E, serum amyloid protein A-1, clusterin, transthyretin, and several albumin fragments were found to be differentially regulated by GH vs. IGF-I in mouse plasma. Thus, we have identified several plasma protein biomarkers that respond specifically and differentially to GH or IGF-I and may represent new physiological targets of these hormones. These findings may lead to better understanding of the independent biological effects of GH vs. IGF-I. In addition, these novel biomarkers may be useful for the development of tests to detect illicit use of GH or IGF-I.

  12. Differential Effects of Growth Hormone Versus Insulin-Like Growth Factor-I on the Mouse Plasma Proteome

    PubMed Central

    Ding, Juan; List, Edward O.; Bower, Brian D.

    2011-01-01

    The GH/IGF-I axis has both pre- and postpubertal metabolic effects. However, the differential effects of GH and/or IGF-I on animal physiology or the plasma proteome are still being unraveled. In this report, we analyzed several physiological effects along with the plasma proteome after treatment of mice with recombinant bovine GH or recombinant human IGF-I. GH and IGF-I showed similar effects in increasing body length, body weight, lean and fluid masses, and organ weights including muscle, kidney, and spleen. However, GH significantly increased serum total cholesterol, whereas IGF-I had no effect on it. Both acute and longer-term effects on the plasma proteome were determined. Proteins found to be significantly changed by recombinant bovine GH and/or recombinant human IGF-I injections were identified by mass spectrometry (MS) and MS/MS. The identities of these proteins were further confirmed by Western blotting analysis. Isoforms of apolipoprotein A4, apolipoprotein E, serum amyloid protein A-1, clusterin, transthyretin, and several albumin fragments were found to be differentially regulated by GH vs. IGF-I in mouse plasma. Thus, we have identified several plasma protein biomarkers that respond specifically and differentially to GH or IGF-I and may represent new physiological targets of these hormones. These findings may lead to better understanding of the independent biological effects of GH vs. IGF-I. In addition, these novel biomarkers may be useful for the development of tests to detect illicit use of GH or IGF-I. PMID:21791560

  13. Two-dimensional proteome reference maps for the soybean cyst nematode Heterodera glycines

    USDA-ARS?s Scientific Manuscript database

    Two-dimensional electrophoresis (2-DE) reference maps of Heterodera glycines were constructed. After in-gel digestion with trypsin, 803 spots representing 426 proteins were subsequently identified by LC-MS/MS. Proteins with annotated function were further categorized by Gene Ontology. Results showed...

  14. Integrated Cellular and Plasma Proteomics of Contrasting B-cell Cancers Reveals Common, Unique and Systemic Signatures.

    PubMed

    Johnston, Harvey E; Carter, Matthew J; Cox, Kerry L; Dunscombe, Melanie; Manousopoulou, Antigoni; Townsend, Paul A; Garbis, Spiros D; Cragg, Mark S

    2017-03-01

    Approximately 800,000 leukemia and lymphoma cases are diagnosed worldwide each year. Burkitt's lymphoma (BL) and chronic lymphocytic leukemia (CLL) are examples of contrasting B-cell cancers; BL is a highly aggressive lymphoid tumor, frequently affecting children, whereas CLL typically presents as an indolent, slow-progressing leukemia affecting the elderly. The B-cell-specific overexpression of the myc and TCL1 oncogenes in mice induce spontaneous malignancies modeling BL and CLL, respectively. Quantitative mass spectrometry proteomics and isobaric labeling were employed to examine the biology underpinning contrasting Eμ-myc and Eμ-TCL1 B-cell tumors. Additionally, the plasma proteome was evaluated using subproteome enrichment to interrogate biomarker emergence and the systemic effects of tumor burden. Over 10,000 proteins were identified (q<0.01) of which 8270 cellular and 2095 plasma proteins were quantitatively profiled. A common B-cell tumor signature of 695 overexpressed proteins highlighted ribosome biogenesis, cell-cycle promotion and chromosome segregation. Eμ-myc tumors overexpressed several methylating enzymes and underexpressed many cytoskeletal components. Eμ-TCL1 tumors specifically overexpressed ER stress response proteins and signaling components in addition to both subunits of the interleukin-5 (IL5) receptor. IL5 treatment promoted Eμ-TCL1 tumor proliferation, suggesting an amplification of IL5-induced AKT signaling by TCL1. Tumor plasma contained a substantial tumor lysis signature, most prominent in Eμ-myc plasma, whereas Eμ-TCL1 plasma contained signatures of immune-response, inflammation and microenvironment interactions, with putative biomarkers in early-stage cancer. These findings provide a detailed characterization of contrasting B-cell tumor models, identifying common and specific tumor mechanisms. Integrated plasma proteomics allowed the dissection of a systemic response and a tumor lysis signature present in early- and late

  15. IgY14 and SuperMix immunoaffinity separations coupled with liquid chromatography-mass spectrometry for human plasma proteomic biomarker discovery

    SciTech Connect

    Shi, Tujin; Zhou, Jianying; Gritsenko, Marina A.; Hossain, Mahmud; Camp, David G.; Smith, Richard D.; Qian, Weijun

    2012-02-01

    Interest in the application of advanced proteomics technologies to human blood plasma- or serum-based clinical samples for the purpose of discovering disease biomarkers continues to grow; however, the enormous dynamic range of protein concentrations in these types of samples (often >10 orders of magnitude) represents a significant analytical challenge, particularly for detecting low-abundance candidate biomarkers. In response, immunoaffinity separation methods for depleting multiple high- and moderate-abundance proteins have become key tools for enriching low-abundance proteins and enhancing detection of these proteins in plasma proteomics. Herein, we describe IgY14 and tandem IgY14-Supermix separation methods for removing 14 high-abundance and up to 60 moderate-abundance proteins, respectively, from human blood plasma and highlight their utility when combined with liquid chromatography-tandem mass spectrometry for interrogating the human plasma proteome.

  16. General intelligence is associated with subclinical inflammation in Nepalese children: A population-based plasma proteomics study.

    PubMed

    Lee, Sun Eun; West, Keith P; Cole, Robert N; Schulze, Kerry J; Wu, Lee Shu-Fune; Yager, James D; Groopman, John; Christian, Parul

    2016-08-01

    Improving child cognition in impoverished countries is a public health priority. Yet, biological pathways and associated biomarkers of impaired cognition remain poorly understood and largely unknown, respectively. This study aimed to explore and quantify associations between functional plasma protein biomarkers and childhood intellectual test performance. We applied proteomics to quantify proteins in plasma samples of 249 rural Nepalese children, 6-8years of age who, 1year later at 7-9years of age, were administered the Universal Nonverbal Intelligence Test (UNIT). Among 751 plasma proteins quantified, 22 were associated with UNIT scores, passing a false discovery rate threshold of 5.0% (q<0.05). UNIT scores were higher by 2.3-9.2 points for every 50% increase in relative abundance of two insulin-like growth factor binding proteins (IGFBPs), six subclasses of apolipoprotein (Apo) and transthyretin, and lower by 4.0-15.3 points for each 50% increase in relative abundance of 13 proteins predominantly involved in inflammation. Among them, IGFBP-acid labile subunit, orosomucoid 1 (ORM1), Apo C-I, and pyruvate kinase isoenzymes M1/M2 jointly explained 37% of the variance in UNIT scores. After additional adjustment for height-for-age Z-score and household socio-economic status as indicators of long-term nutritional and social stress, associations with 6 proteins involved in inflammation, including ORM1, α-1-antichymotrypsin, reticulocalbin 1, and 3 components of the complement cascade, remained significant (q<0.05). Using untargeted proteomics, stable, constitutive facets of subclinical inflammation were associated with lower developmental test performance in this rural South Asian child population. Plasma proteomics may offer opportunities to identify functional, antecedent biomarkers of child cognitive development.

  17. Design and characterization of an RF excited micro atmospheric pressure plasma jet for reference in plasma medicine

    NASA Astrophysics Data System (ADS)

    Schulz-von der Gathen, Volker

    2015-09-01

    Over the last decade a huge variety of atmospheric pressure plasma jets has been developed and applied for plasma medicine. The efficiency of these non-equilibrium plasmas for biological application is based on the generated amounts of reactive species and radiation. The gas temperatures stay within a range tolerable for temperature-sensitive tissues. The variety of different discharge geometries complicates a direct comparison. In addition, in plasma-medicine the combination of plasma with reactive components, ambient air, as well as biologic tissue - typically also incorporating fluids - results in a complex system. Thus, real progress in plasma-medicine requires a profound knowledge of species, their fluxes and processes hitting biological tissues. That will allow in particular the necessary tailoring of the discharge to fit the conditions. The complexity of the problem can only be overcome by a common effort of many groups and requires a comparison of their results. A reference device based on the already well-investigated micro-scaled atmospheric pressure plasma jet is presented. It is developed in the frame of the European COST initiative MP1101 to establish a publicly available, stable and reproducible source, where required plasma conditions can be investigated. Here we present the design and the ideas behind. The presentation discusses the requirements for the reference source and operation conditions. Biological references are also defined by the initiative. A specific part of the talk will be attributed to the reproducibility of results from various samples of the device. Funding by the DFG within the Package Project PAK816 ``Plasma Cell Interaction in Dermatology'' and the Research Unit FOR 1123 ``Physics of microplasmas'' is gratefully acknowledged.

  18. Proteomic Changes of Tissue-Tolerable Plasma Treated Airway Epithelial Cells and Their Relation to Wound Healing

    PubMed Central

    Lendeckel, Derik; Eymann, Christine; Emicke, Philipp; Daeschlein, Georg; Darm, Katrin; O'Neil, Serena; Beule, Achim G.; von Woedtke, Thomas; Völker, Uwe; Weltmann, Klaus-Dieter; Jünger, Michael; Hosemann, Werner; Scharf, Christian

    2015-01-01

    Background. The worldwide increasing number of patients suffering from nonhealing wounds requires the development of new safe strategies for wound repair. Recent studies suggest the possibility of nonthermal (cold) plasma application for the acceleration of wound closure. Methods. An in vitro wound healing model with upper airway S9 epithelial cells was established to determine the macroscopically optimal dosage of tissue-tolerable plasma (TTP) for wound regeneration, while a 2D-difference gel electrophoresis (2D-DIGE) approach was used to quantify the proteomic changes in a hypothesis-free manner and to evaluate the balance of beneficial and adverse effects due to TTP application. Results. Plasma doses from 30 s up to 360 s were tested in relation to wound closure after 24 h, 48 h, 72 h, 96 h, and 120 h, in which lower doses (30, 60, and 120 s) resulted in dose-dependent improved wound healing rate compared to untreated cells. Thereby, the 120 s dose caused significantly the best wound healing properties after 96 and 120 h. The proteome analysis combined with IPA revealed that a lot of affected stress adaptation responses are linked to oxidative stress response emphasizing oxidative stress as a possible key event in the regeneration process of epithelial cells as well as in the adaptation to plasma exposure. Further cellular and molecular functions like proliferation and apoptosis were significantly up- or downregulated by all TTP treatments but mostly by the 120 s dose. Conclusions. For the first time, we were able to show plasma effects on cellular adaptation of upper airway epithelial S9 cells improving wound healing. This is of particular interest for plasma application, for example, in the surgery field of otorhinolaryngology or internal medicine. PMID:26539504

  19. Quantitative proteomic analysis by iTRAQ for identification of candidate biomarkers in plasma from acute respiratory distress syndrome patients.

    PubMed

    Chen, Xia; Shan, Qiang; Jiang, Li; Zhu, Bo; Xi, Xiuming

    2013-11-08

    Acute respiratory distress syndrome (ARDS) is a major cause of morbidity and mortality in critical patients. Proteomic analysis of plasma from individuals with ARDS could elucidate new biomarkers for diagnosis and pathophysiology and identify potential ARDS treatment targets. In this study, we recruited 26 patients (15 controls, 11 ARDS). The ARDS group was subdivided into two groups depending on the type of injury: (1) direct lung injury (AD) and (2) indirect lung injury (AI). Using iTRAQ (isobaric tags for relative and absolute quantitation) analysis, we identified 2429 peptides representing 132 plasma proteins. Among these, 16 were differentially expressed in ARDS patients, including 11 overlapping proteins between the AI and AD group and 5 AI-specific proteins. Protein annotation revealed that lipid transport and complement activation were significantly enriched in the biological process category, and lipid transporter, transporter, and serine-type peptidase activities were significantly enriched in the molecular function category. IPA (Ingenuity Pathway Analysis) signaling pathways revealed that the overlapping proteins were involved in a variety of signaling pathways, including those underlying acute phase response; liver X receptor/retinoid X receptor (LXR/RXR) and farnesoid X (FXR)/RXR activation; clathrin-mediated endocytosis; atherosclerosis; interleukin (IL)-12; complement system; and cytokine, nitric oxide, and reactive oxygen species production in macrophages. We present the first proteomic analysis of ARDS plasma using the iTRAQ approach. Our data provide new biomarker candidates and shed light on potential pathological mechanisms underlying ARDS.

  20. Comparative analysis of Brassica napus plasma membrane proteins under phosphorus deficiency using label-free and MaxQuant-based proteomics approaches.

    PubMed

    Chen, Shuisen; Luo, Ying; Ding, Guangda; Xu, Fangsen

    2016-02-05

    Phosphorus (P) deficiency is a primary constraint for plant growth in terrestrial ecosystems. To better understand the genotypic differences in the adaptation mechanism of Brassica napus to P deficiency, we purified the plasma membrane (PM) from the roots of two genotypes: P-efficient "Eyou Changjia" and P-inefficient "B104-2". Combining label-free quantitative proteomics with the MaxQuant approach, a total of 71 proteins that significantly changed in abundances were identified in the two genotypes in response to P-free starvation, including 31 in "Eyou Changjia" and 40 in "B104-2". Based on comparative genomics study, 28 proteins were mapped to the confidence intervals of quantitative trait loci (QTLs) for P efficiency related traits. Seven decreased proteins with transporter activity were found to be located in the PM by subcellular localization analyses. These proteins involved in intracellular protein transport and ATP hydrolysis coupled proton transport were mapped to the QTL for P content and dry weight. Compared with "B104-2", more decreased proteins referring to transporter activity were found in "Eyou Changjia", showing that substance exchange was decreased in response to short-term P-free starvation. Together with the finding, more decreased proteins functioning in signal transduction and protein synthesis/degradation suggested that "Eyou Changjia" could slow the progression of growth and save more P in response to short-term P-free starvation. P deficiency seriously limits the production and quality of B. napus. Roots absorb water and nutrients and anchor the plant in the soil. Therefore, to study root PM proteome under P stress would be helpful to understand the adaptation mechanism for P deficiency. However, PM proteome analysis in B. napus has been seldom reported due to the high hydrophobicity and low abundance of PM. Thus, we herein investigated the PM proteome alteration of roots in two B. napus genotypes, with different P deficient tolerances, in

  1. Whole-cell proteome reference maps of an extreme thermophile, Thermus thermophilus HB8.

    PubMed

    Kim, Kwang; Okanishi, Hiroki; Masui, Ryoji; Harada, Akira; Ueyama, Norikazu; Kuramitsu, Seiki

    2012-10-01

    Thermus thermophilus HB8 is a model microorganism for industrial applications because of its thermophilic enzymes, and for basic bacteriology to understand the coordination of the biological functions of the genome-encoded enzymes at the cellular level. Here, we present 2DE reference maps of T. thermophilus HB8 in the pH ranges 4-7 and 6-11 obtained with whole-cell lysates. PMF analysis using MALDI-TOF-MS and MS/MS analysis using nano-scale LC and quadrupole TOF-MS identified 258 different proteins among the 306 protein spots on 2DE gels. Functional classification indicated that 56%, 16%, and 14% of the identified proteins were related to metabolism, genetic information process, and cellular process, respectively. Detailed classification of the metabolism-related proteins suggested that during the exponential phase, amino acid and carbohydrate metabolism are major metabolic processes, whereas nucleotide and lipid metabolism are minor ones. On the other hand, volume quantification analysis revealed that proteins involved in the translational process, nucleotide metabolism, and central carbon metabolism were most abundantly expressed in the exponential phase. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Effects of preanalytical variables on peptide and protein measurements in human serum and plasma: implications for clinical proteomics.

    PubMed

    Rai, Alex J; Vitzthum, Frank

    2006-08-01

    There is a wealth of knowledge in the field of in vitro diagnostics with regard to preanalytical variables and their impact on the determination of peptide and protein analytes in human serum and plasma. This information is applicable to clinical proteomics investigations, which utilize the same sample types. Studies have demonstrated that the majority of variations and errors in in vitro diagnostics seem to occur in the preanalytical phase prior to specimen analysis. Preanalytical processes include study design, compliance of the subjects investigated, compliance of the technical staff in adherence to protocols, choice of specimens utilized and sample collection and processing. These variables can have a dramatic impact on the determination of analytes and can affect result outcomes, reproducibility and the validity of investigations. By drawing analogies to in vitro diagnostics practices, specific variables that are likely to impact the results of proteomics studies can be identified. Recognition of such variables is the first step towards their understanding and, eventually, controlling their impact. In this article, we will review preanalytical variables, provide examples for their effects on the determination of distinct peptides and proteins and discuss potential implications for clinical proteomics investigations.

  3. Proteomic Investigation on Grp94-IgG Complexes Circulating in Plasma of Type 1 Diabetic Subjects

    PubMed Central

    Roveri, Antonella; Zaccarin, Mattia; Pagetta, Andrea; Tramentozzi, Elisa; Finotti, Paola

    2015-01-01

    The glucose-regulated protein94 (Grp94) has been found in complexes with IgG in plasma of Type 1 (T1) diabetic subjects; however, the pathogenetic meaning of Grp94-IgG complexes has not yet been elucidated. To shed light on the nature and structure of these complexes in vivo, we conducted a proteomic analysis on plasma of both T1 diabetic subjects and healthy control subjects. IgG purified from plasma was submitted to 2D PAGE followed by Western blotting and mass analysis. Grp94 was detected in plasma of all diabetic but not control subjects and found linked with its N-terminus to the IgG heavy chain. Mass analysis of heavy chain of IgG that binds Grp94 also in vitro, forming stable complexes with characteristics similar to those of native ones, permitted identifying CH2 and CH3 regions as those involved in binding Grp94. At the electron microscopy, IgG from diabetic plasma appeared as fibrils of various lengthes and dimensions, suggestive of elevated aggregating tendency conferred to IgG by Grp94. The nonimmune nature of complexes turned out to be responsible for the particular stability and structure adopted by complexes in plasma of diabetic subjects. Results are of relevance to understanding the pathogenetic mechanisms underlying diabetes and its complications. PMID:26167512

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

    PubMed

    Byrum, Stephanie D; Burdine, Marie S; Orr, Lisa; Mackintosh, Samuel G; Authier, Simon; Pouliot, Mylene; Hauer-Jensen, Martin; Tackett, Alan J

    2017-01-01

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

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

    PubMed Central

    Burdine, Marie S.; Orr, Lisa; Mackintosh, Samuel G.; Authier, Simon; Pouliot, Mylene; Hauer-Jensen, Martin; Tackett, Alan J.

    2017-01-01

    Acute radiation syndrome (ARS) is a complex multi-organ disease resulting from total body exposure to high doses of radiation. Individuals can be exposed to total body irradiation (TBI) in a number of ways, including terrorist radiological weapons or nuclear accidents. In order to determine whether an individual has been exposed to high doses of radiation and needs countermeasure treatment, robust biomarkers are needed to estimate radiation exposure from biospecimens such as blood or urine. In order to identity such candidate biomarkers of radiation exposure, high-resolution proteomics was used to analyze plasma from non-human primates following whole body irradiation (Co-60 at 6.7 Gy and 7.4 Gy) with a twelve day observation period. A total of 663 proteins were evaluated from the plasma proteome analysis. A panel of plasma proteins with characteristic time- and dose-dependent changes was identified. In addition to the plasma proteomics study reported here, we recently identified candidate biomarkers using urine from these same non-human primates. From the proteomic analysis of both plasma and urine, we identified ten overlapping proteins that significantly differentiate both time and dose variables. These shared plasma and urine proteins represent optimal candidate biomarkers of radiation exposure. PMID:28350824

  6. Functional and proteomic alterations of plasma high density lipoproteins in type 1 diabetes mellitus.

    PubMed

    Manjunatha, Shankarappa; Distelmaier, Klaus; Dasari, Surendra; Carter, Rickey E; Kudva, Yogish C; Nair, K Sreekumaran

    2016-09-01

    Higher HDL-cholesterol (HDL-C) is linked to lower cardiovascular risk but individuals with type 1 diabetes mellitus (T1DM) with normal or high HDL-C have higher cardiovascular events compared to age matched non-diabetic controls (ND). We determined whether altered HDL functions despite having normal HDL-C concentration may explain increased cardiovascular risk in T1DM individuals. We also determined whether irreversible posttranslational modifications (PTMs) of HDL bound proteins occur in T1DM individuals with altered HDL functions. T1DM with poor glycemic control (T1D-PC, HbA1c≥8.5%, n=15) and T1DM with good glycemic control (T1D-GC, HbA1c≤6.6%, n=15) were compared with equal numbers of NDs, ND-PC and ND-GC respectively, matched for age, sex and body mass index (BMI). We measured cholesterol efflux capacity (CEC) of HDL in the serum using J774 macrophages, antioxidant function of HDL as the ability to reverse the oxidative damage of LDL and PON1 activity using commercially available kit. For proteomic analysis, HDL was isolated by density gradient ultracentrifugation and was analyzed by mass spectrometry and shotgun proteomics method. Plasma HDL-C concentrations in both T1DM groups were similar to their ND. However, CEC (%) of T1D-PC (16.9±0.8) and T1D-GC (17.1±1) were lower than their respective ND (17.9±1, p=0.01 and 18.2±1.4, p=0.02). HDL antioxidative function also was lower (p<0.05). The abundance of oxidative PTMs of apolipoproteins involved in CEC and antioxidative functions of HDL were higher in T1D-PC (ApoA4, p=0.041) and T1D-GC (ApoA4, p=0.025 and ApoE, p=0.041) in comparison with ND. Both T1D-PC and T1D-GC groups had higher abundance of amadori modification of ApoD (p=0.002 and p=0.041 respectively) and deamidation modification of ApoA4 was higher in T1D-PC (p=0.025). Compromised functions of HDL particles in T1DM individuals, irrespective of glycemic control, could be explained by higher abundance of irreversible PTMs of HDL proteins. These

  7. Reference Intervals for Plasma Amyloid β in Korean Adults Without Cognitive Impairment.

    PubMed

    Kim, Min Young; Kim, Kyu Nam; Cho, Hye Min; Lee, Duck Joo; Cho, Doo Yeoun

    2016-11-01

    Amyloid β (Aβ) peptides are important components of plaques in patients with Alzheimer's disease (AD). Recent studies suggest that a low plasma ratio of Aβ42 to Aβ40 may precede the development of the sporadic form of AD. The aim of this study was to establish reference intervals for plasma Aβ in Korean adults. A total of 370 apparently healthy individuals (181 males and 189 females aged 40-69 yr) without cognitive impairment were enrolled. Plasma concentrations of Aβ40 and Aβ42 were measured by using a human amyloid β assay kit (Immuno-Biological Laboratories, Japan). Reference intervals were established according to the "CLSI guidelines for defining, establishing, and verifying reference intervals in the clinical laboratory". There was no need to partition the data with respect to gender or age group. The 95th percentile reference intervals for Aβ40 and Aβ42 were 127-331 pg/mL and 2.31-19.84 pg/mL, respectively. The reference interval for the Aβ42/Aβ40 ratio was 0.011-0.092. Plasma Aβ concentrations obtained in this study could be used as reference intervals for clinical purposes.

  8. [Search for potential gastric cancer biomarkers using low molecular weight blood plasma proteome profiling by mass spectrometry].

    PubMed

    Shevchenko, V E; Arnotskaia, N E; Ogorodnikova, E V; Davydov, M M; Ibraev, M A; Turkin, I N; Davydov, M I

    2014-01-01

    Gastric cancer, one of the most widespread malignant tumors, still lacks reliable serum/plasma biomarkers of its early detection. In this study we have developed, unified, and tested a new methodology for search of gastric cancer biomarkers based on profiling of low molecular weight proteome (LMWP) (1-17 kDa). This approach included three main components: sample pre-fractionation, matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), data analysis by a bioinformatics software package. Applicability and perspectives of the developed approach for detection of potential gastric cancer markers during LMWP analysis have been demonstrated using 69 plasma samples from patients with gastric cancer (stages I-IV) and 238 control samples. The study revealed peptides/polypeptides, which may be potentially used for detection of this pathology.

  9. Plasma Proteomic Signature in Overweight Girls Closely Correlates with Homeostasis Model Assessment (HOMA), an Objective Measure of Insulin Resistance

    PubMed Central

    Poth, Merrily; McIver, Harkirtin; Ayika, Chiedozie; Eidelman, Ofer; Jozwik, Catherine; Pollard, Harvey B.

    2011-01-01

    Obesity is known to be associated with a large number of long-term morbidities, and while in some cases the relationship of obesity and the consequences is clear (for example, excess weight and lower extremity orthopedic problems) in others the mechanism is not as clear. One common system of categorizing overweight in terms of the likelihood of negative consequences involves using the concept of “metabolic syndrome”. We hypothesized that the development of a plasma protein profile of overweight adolescents with and without the metabolic syndrome might give a more precise and informative picture of the disease process than the current clinical categorization and permit early targeted intervention. For this paper, we used antibody microarrays to analyze the plasma proteome of a group of 15 overweight female adolescent patients. Upon analysis of the proteome, the overweight patients diverged from the nonoverweight female controls. Furthermore, the overweight patients were divided by the analysis into two population clusters, each with distinctive protein expression patterns. Interestingly, the clusters were characterized by differences in insulin resistance, as measured by HOMA. Categorization according to the presence or absence of the metabolic syndrome did not yield such clusters. PMID:22442648

  10. Investigation of Plasma Biomarkers in HIV-1/HCV Mono- and Coinfected Individuals by Multiplex iTRAQ Quantitative Proteomics

    PubMed Central

    Shetty, Vivekananda; Jain, Pooja; Nickens, Zacharie; Sinnathamby, Gomathinayagam; Mehta, Anand

    2011-01-01

    Abstract The analysis of plasma samples from HIV-1/HCV mono- and coinfected individuals by quantitative proteomics is an efficient strategy to investigate changes in protein abundances and to characterize the proteins that are the effectors of cellular functions involved in viral pathogenesis. In this study, the infected and healthy plasma samples (in triplicate) were treated with ProteoMiner beads to equalize protein concentrations and subjected to 4-plex iTRAQ labeling and liquid chromatography/mass spectrometry (LC-MS/MS) analysis. A total of 70 proteins were identified with high confidence in the triplicate analysis of plasma proteins and 65% of the proteins were found to be common among the three replicates. Apolipoproteins and complement proteins are the two major classes of proteins that exhibited differential regulation. The results of quantitative analysis revealed that APOA2, APOC2, APOE, C3, HRG proteins were upregulated in the plasma of all the three HIV-1 mono-, HCV mono-, and coinfected patient samples compared to healthy control samples. Ingenuity pathway analysis (IPA) of the upregulated proteins revealed that they are implicated in the hepatic lipid metabolism, inflammation, and acute-phase response signaling pathways. Thus, we identified several differentially regulated proteins in HIV-1/HCV mono and coinfected plasma samples that may be potential biomarkers for liver disease. PMID:21978398

  11. Plasma Proteome Dynamics: Analysis of Lipoproteins and Acute Phase Response Proteins with 2H2O Metabolic Labeling*

    PubMed Central

    Li, Ling; Willard, Belinda; Rachdaoui, Nadia; Kirwan, John P.; Sadygov, Rovshan G.; Stanley, William C.; Previs, Stephen; McCullough, Arthur J.; Kasumov, Takhar

    2012-01-01

    Understanding the pathologies related to the regulation of protein metabolism requires methods for studying the kinetics of individual proteins. We developed a 2H2O metabolic labeling technique and software for protein kinetic studies in free living organisms. This approach for proteome dynamic studies requires the measurement of total body water enrichments by GC-MS, isotopic distribution of the tryptic peptide by LC-MS/MS, and estimation of the asymptotical number of deuterium incorporated into a peptide by software. We applied this technique to measure the synthesis rates of several plasma lipoproteins and acute phase response proteins in rats. Samples were collected at different time points, and proteins were separated by a gradient gel electrophoresis. 2H labeling of tryptic peptides was analyzed by ion trap tandem mass spectrometry (LTQ MS/MS) for measurement of the fractional synthesis rates of plasma proteins. The high sensitivity of LTQ MS in zoom scan mode in combination with 2H label amplification in proteolytic peptides allows detection of the changes in plasma protein synthesis related to animal nutritional status. Our results demonstrate that fasting has divergent effects on the rate of synthesis of plasma proteins, increasing synthesis of ApoB 100 but decreasing formation of albumin and fibrinogen. We conclude that this technique can effectively measure the synthesis of plasma proteins and can be used to study the regulation of protein homeostasis under physiological and pathological conditions. PMID:22393261

  12. Novel multifunctional chitosan-GMA-IDA-Cu(II) nanospheres for high dynamic range characterization of the human plasma proteome.

    PubMed

    Zou, Xiajuan; Zhong, Lijun; Liu, Dan; Yang, Bin; Lou, Yaxin; Peng, Jiarou; Rainer, Matthias; Feuerstein, Isabel; Muhammad, Najam-ul-Haq; Huck, Christian W; Bonn, Günther K; Yin, Yuxin

    2011-05-01

    In this study, we describe characterization of the human plasma proteome based on analysis with multifunctional chitosan-GMA-IDA-Cu(II) nanospheres. Chitosan-GMA-IDA-Cu(II) nanospheres with diameters of 20 to 100 nm have unique properties due to multifunctional chemical moieties, high surface area, high capacity, good dispersibility in buffer solution as well as good biocompatibility and chemical stability which improves their specific interaction with peptides and proteins of the human plasma using different binding buffers. Combining these chitosan-GMA-IDA-Cu(II) nanospheres with MS spectrometry results in a novel strategy which should make it possible to characterize the plasma proteome in a single test. Peptides and proteins adsorbed on the nanosphere can be directly detected by MALDI-TOF-MS. The eluted lower molecular weight peptides and proteins are identified by nano-LC-ESI-MS/MS. A total of 842 unique LMW peptides and 1,682 human unredundant proteins IDs were identified in two different binding buffers, which included relatively low-level proteins (e.g., pg/mL of IL3 Interleukin-3) co-distributed with high-abundance proteins (e.g., 35-55 mg/mL level serum albumin). As such, this nanosphere technique selectively enabled the identification of proteins over a dynamic range of greater than 8 orders of magnitude. Considering this capacity for selective enrichment of peptides and proteins in human plasma, and the large number of LMW peptides and proteins which can be identified, this method promises to accelerate discovery of biomarkers for clinical application.

  13. The maternal plasma proteome changes as a function of gestational age in normal pregnancy: a longitudinal study.

    PubMed

    Romero, Roberto; Erez, Offer; Maymon, Eli; Chaemsaithong, Piya; Xu, Zhonghui; Pacora, Percy; Chaiworapongsa, Tinnakorn; Done, Bogdan; Hassan, Sonia S; Tarca, Adi L

    2017-07-01

    Pregnancy is accompanied by dramatic physiological changes in maternal plasma proteins. Characterization of the maternal plasma proteome in normal pregnancy is an essential step for understanding changes to predict pregnancy outcome. The objective of this study was to describe maternal plasma proteins that change in abundance with advancing gestational age and determine biological processes that are perturbed in normal pregnancy. A longitudinal study included 43 normal pregnancies that had a term delivery of an infant who was appropriate for gestational age without maternal or neonatal complications. For each pregnancy, 3 to 6 maternal plasma samples (median, 5) were profiled to measure the abundance of 1125 proteins using multiplex assays. Linear mixed-effects models with polynomial splines were used to model protein abundance as a function of gestational age, and the significance of the association was inferred via likelihood ratio tests. Proteins considered to be significantly changed were defined as having the following: (1) >1.5-fold change between 8 and 40 weeks of gestation; and (2) a false discovery rate-adjusted value of P < .1. Gene ontology enrichment analysis was used to identify biological processes overrepresented among the proteins that changed with advancing gestation. The following results were found: (1) Ten percent (112 of 1125) of the profiled proteins changed in abundance as a function of gestational age; (2) of the 1125 proteins analyzed, glypican-3, sialic acid-binding immunoglobulin-type lectin-6, placental growth factor, C-C motif-28, carbonic anhydrase 6, prolactin, interleukin-1 receptor 4, dual-specificity mitogen-activated protein kinase 4, and pregnancy-associated plasma protein-A had more than a 5-fold change in abundance across gestation (these 9 proteins are known to be involved in a wide range of both physiological and pathological processes, such as growth regulation, embryogenesis, angiogenesis immunoregulation, inflammation

  14. Unilateral and bilateral adrenalectomy for pheochromocytoma requires adjustment of urinary and plasma metanephrine reference ranges.

    PubMed

    Osinga, Thamara E; van den Eijnden, Maartje H A; Kema, Ido P; Kerstens, Michiel N; Dullaart, Robin P F; de Jong, Wilhelmina H A; Sluiter, Wim J; Links, Thera P; van der Horst-Schrivers, Anouk N A

    2013-03-01

    Follow-up after adrenalectomy for pheochromocytoma is recommended because of a recurrence risk. During follow-up, plasma and/or urinary metanephrine (MN) and normetanephrine (NMN) are interpreted using reference ranges obtained in healthy subjects. Because adrenalectomy may decrease epinephrine production, we compared MN and NMN concentrations in patients after adrenalectomy to concentrations in a healthy reference population. A single-center cohort study was performed in pheochromocytoma patients after adrenalectomy between 1980 and 2011. Seventy patients after unilateral and 24 after bilateral adrenalectomy were included. Plasma-free and urinary-deconjugated MN and NMN determined at 3 to 6 months and annually until 5 years after adrenalectomy were compared with concentrations in a reference population. Data are presented in median (interquartile range). Urinary and plasma MN concentrations 3 to 6 months after unilateral adrenalectomy were lower compared with the reference population (39 [31-53] μmol/mol creatinine and 0.14 [0.09-0.18] nmol/L vs 61 [49-74] μmol/mol creatinine and 0.18 [0.13-0.23] nmol/L, respectively, both P < .05). Urinary MN after bilateral adrenalectomy was reduced even further (7 [1-22] μmol/mol creatinine; P < .05). Urinary and plasma NMN were higher after unilateral adrenalectomy (151 [117-189] μmol/mol creatinine and 0.78 [0.59-1.00] nmol/L vs 114 [98-176] μmol/mol creatinine and 0.53 [0.41-0.70] nmol/L; both P < .05). Urinary NMN after bilateral adrenalectomy was higher (177 [106-238] μmol/mol creatinine; P < .05). Changes in urinary and plasma MNs persisted during follow-up. Concentrations of MN are decreased, whereas NMN concentrations are increased after unilateral and bilateral adrenalectomy. Adjusted reference values for MN and NMN are needed in the postsurgical follow-up of pheochromocytoma patients.

  15. Langmuir Probe Measurements in an Inductively Coupled GEC Reference Cell Plasma

    NASA Technical Reports Server (NTRS)

    Ji, J. S.; Kim, J. S.; Cappelli, M. A.; Sharma, S. P.; Arnold, J. O. (Technical Monitor)

    1998-01-01

    Measurements of electron number density, electron temperature, and electron energy distribution function (EEDF) using a compensated Langmuir probe have been performed on an inductively (transformer ) coupled Gaseous Electronics Conference (GEC) reference cell plasma. The plasma source is operated with CH4, CF4, or their mixtures with argon. The effect of independently driving the electrode supporting the wafer on the probe data is studied. In particular, we find that the plasma structure depends on the phase in addition to the magnitude of the power coupled to the electrode relative to that of the transformer coil. The Langmuir probe is translated in a plane parallel to the electrode to investigate the spatial structure of the plasma. The probe data is also compared with fluid model predictions.

  16. A reference protocol for comparing the biocidal properties of gas plasma generating devices

    NASA Astrophysics Data System (ADS)

    Shaw, A.; Seri, P.; Borghi, C. A.; Shama, G.; Iza, F.

    2015-12-01

    Growing interest in the use of non-thermal, atmospheric pressure gas plasmas for decontamination purposes has resulted in a multiplicity of plasma-generating devices. There is currently no universally approved method of comparing the biocidal performance of such devices and in the work described here spores of the Gram positive bacterium Bacillus subtilis (ATCC 6633) are proposed as a suitable reference biological agent. In order to achieve consistency in the form in which the biological agent in question is presented to the plasma, a polycarbonate membrane loaded with a monolayer of spores is proposed. The advantages of the proposed protocol are evaluated by comparing inactivation tests in which an alternative microorganism (methicillin resistant Staphylococcus aureus—MRSA) and the widely-used sample preparation technique of directly pipetting cell suspensions onto membranes are employed. In all cases, inactivation tests with either UV irradiation or plasma exposure were more reproducible when the proposed protocol was followed.

  17. A comparative proteomics method for multiple samples based on a (18)O-reference strategy and a quantitation and identification-decoupled strategy.

    PubMed

    Wang, Hongbin; Zhang, Yongqian; Gui, Shuqi; Zhang, Yong; Lu, Fuping; Deng, Yulin

    2017-08-15

    Comparisons across large numbers of samples are frequently necessary in quantitative proteomics. Many quantitative methods used in proteomics are based on stable isotope labeling, but most of these are only useful for comparing two samples. For up to eight samples, the iTRAQ labeling technique can be used. For greater numbers of samples, the label-free method has been used, but this method was criticized for low reproducibility and accuracy. An ingenious strategy has been introduced, comparing each sample against a (18)O-labeled reference sample that was created by pooling equal amounts of all samples. However, it is necessary to use proportion-known protein mixtures to investigate and evaluate this new strategy. Another problem for comparative proteomics of multiple samples is the poor coincidence and reproducibility in protein identification results across samples. In present study, a method combining (18)O-reference strategy and a quantitation and identification-decoupled strategy was investigated with proportion-known protein mixtures. The results obviously demonstrated that the (18)O-reference strategy had greater accuracy and reliability than other previously used comparison methods based on transferring comparison or label-free strategies. By the decoupling strategy, the quantification data acquired by LC-MS and the identification data acquired by LC-MS/MS are matched and correlated to identify differential expressed proteins, according to retention time and accurate mass. This strategy made protein identification possible for all samples using a single pooled sample, and therefore gave a good reproducibility in protein identification across multiple samples, and allowed for optimizing peptide identification separately so as to identify more proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Differential proteomic approach for identification and verification of aberrantly glycosylated proteins in adenocarcinoma lung cancer (ADLC) plasmas by lectin-capturing and targeted mass spectrometry.

    PubMed

    Ahn, Yeong Hee; Ji, Eun Sun; Oh, Na Ree; Kim, Yong-Sam; Ko, Jeong Heon; Yoo, Jong Shin

    2014-06-25

    To investigate quantitative differences in aberrant glycosylation of target glycoproteins between noncancerous group and patient group with adenocarcinoma lung cancer (ADLC), differential proteomic approach was developed by cooperatively using comparative lectin-capturing, targeted mass spectrometry (MRM MS), and antibody/lectin sandwich ELISA. Plasma samples comparatively prepared from 3 ADLC patients and 3 controls, with and without lectin-fractionation using fucose-specific Aleuria aurantia lectin (AAL), were trypsin-digested and analyzed for target glycoproteins, alpha-1-acid glycoprotein (AGP) and ceruloplasmin (CP), by MRM MS. From the MRM MS data the abundance levels of AAL-captured glycoforms of both targets were significantly higher in ADLC cases compared to controls, although the levels in total protein abundance were comparable between ADLC and control groups. This difference between ADLC and control groups in the fucosylated glycoform levels was originated mainly from aberrant fucosylation on the targets in ADLC plasmas rather than change in total protein abundance of the targets, and also confirmed by sandwich ELISA. AGP and CP were further verified to be biomarker candidates by MRM-based analysis of AAL-captured plasmas (30 ADLC cases, 30 controls), with AUROC 0.758 and 0.847 respectively. This differential proteomic approach can be useful for identifying and verifying biomarker candidate involved in aberrant protein glycosylation. The present paper introduces an efficient differential proteomic method to investigate quantitative differences in aberrant protein glycosylation of serological glycoproteins between noncancerous group and lung cancer patient group. This differential proteomic approach consisting of the targeted MRM MS of comparatively lectin-captured plasma fractions and the antibody/lectin sandwich ELISA-based assay was evaluated to be useful for identification of aberrantly fucosylated glycoproteins AGP and CP in lung cancer plasmas. In

  19. Novel alternative splicing isoform biomarkers identification from high-throughput plasma proteomics profiling of breast cancer

    PubMed Central

    2013-01-01

    Background In the biopharmaceutical industry, biomarkers define molecular taxonomies of patients and diseases and serve as surrogate endpoints in early-phase drug trials. Molecular biomarkers can be much more sensitive than traditional lab tests. Discriminating disease biomarkers by traditional method such as DNA microarray has proved challenging. Alternative splicing isoform represents a new class of diagnostic biomarkers. Recent scientific evidence is demonstrating that the differentiation and quantification of individual alternative splicing isoforms could improve insights into disease diagnosis and management. Identifying and characterizing alternative splicing isoforms are essential to the study of molecular mechanisms and early detection of complex diseases such as breast cancer. However, there are limitations with traditional methods used for alternative splicing isoform determination such as transcriptome-level, low level of coverage and poor focus on alternative splicing. Results Therefore, we presented a peptidomics approach to searching novel alternative splicing isoforms in clinical proteomics. Our results showed that the approach has significant potential in enabling discovery of new types of high-quality alternative splicing isoform biomarkers. Conclusions We developed a peptidomics approach for the proteomics community to analyze, identify, and characterize alternative splicing isoforms from MS-based proteomics experiments with more coverage and exclusive focus on alternative splicing. The approach can help generate novel hypotheses on molecular risk factors and molecular mechanisms of cancer in early stage, leading to identification of potentially highly specific alternative splicing isoform biomarkers for early detection of cancer. PMID:24565027

  20. Proteome Changes in the Plasma of Myelodysplastic Syndrome Patients with Refractory Anemia with Excess Blasts Subtype 2

    PubMed Central

    Majek, Pavel; Riedelova-Reicheltova, Zuzana; Pecankova, Klara; Cermak, Jaroslav; Dyr, Jan E.

    2014-01-01

    The goal of this study was to explore the plasma proteome of myelodysplastic syndrome (MDS) patients with refractory anemia with excess blasts subtype 2 (RAEB-2) in comparison to healthy controls. 20 plasma samples were separated with 2D electrophoresis and statistically processed with Progenesis SameSpots software. 47 significantly differing (P < 0.05) spots were observed, and 27 different proteins were identified by nano-LC-MS/MS. Mass spectrometry-based relative label-free quantification showed a 2-fold increase of the leucine-rich alpha-2-glycoprotein (LRAG) peptide levels in the RAEB-2 group. Changes in the fragments of the inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4) protein were observed. Western blot analysis showed no differences in albumin and ITIH4 levels, while increased expression was observed for LRAG in the RAEB-2 group. Quantification using ELISA showed decreased plasma level of alpha-2-HS glycoprotein in the RAEB-2 group. In conclusion, this is the first time that alpha-2-HS glycoprotein and LRAG were proposed as new biomarkers of RAEB-2 and advanced MDS, respectively. Alpha-2-HS glycoprotein, a protein involved in the bone marrow development and previously proposed as a MDS biomarker candidate, was significantly decreased in RAEB-2. Increased expression and changes in modification(s) were observed for LRAG, a protein involved in granulocytic and neutrophil differentiation, and angiogenesis. PMID:24958999

  1. Label-Free Proteome Analysis of Plasma from Patients with Breast Cancer: Stage-Specific Protein Expression

    PubMed Central

    Lobo, Marina Duarte Pinto; Moreno, Frederico Bruno Mendes Batista; Souza, Gustavo Henrique Martins Ferreira; Verde, Sara Maria Moreira Lima; Moreira, Renato de Azevedo; Monteiro-Moreira, Ana Cristina de Oliveira

    2017-01-01

    Breast cancer is one of the most commonly diagnosed types of cancer among women. Breast cancer mortality rates remain high probably because its diagnosis is hampered by inaccurate detection methods. Since changes in protein expression as well as modifications in protein glycosylation have been frequently reported in cancer development, the aim of this work was to study the differential expression as well as modifications of glycosylation of proteins from plasma of women with breast cancer at different stages of disease (n = 30) compared to healthy women (n = 10). A proteomics approach was used that depleted albumin and IgG from plasma followed by glycoprotein enrichment using immobilized Moraceae lectin (frutalin)-affinity chromatography and data-independent label-free mass spectrometric analysis. Data are available via ProteomeXchange with identifier PXD003106. As result, 57,016 peptides and 4,175 proteins among all samples were identified. From this, 40 proteins present in unbound (PI—proteins that did not interact with lectin) and bound (PII—proteins that interacted with lectin) fractions were differentially expressed. High levels of apolipoprotein A-II were detected here that were elevated significantly in the early and advanced stages of the disease. Apolipoprotein C-III was detected in both fractions, and its level was increased slightly in the PI fraction of patients with early-stage breast cancer and expressed at higher levels in the PII fraction of patients with early and intermediate stages. Clusterin was present at higher levels in both fractions of patients with early and intermediate stages of breast cancer. Our findings reveal a correlation between alterations in protein glycosylation, lipid metabolism, and the progression of breast cancer. PMID:28210565

  2. Tokamak magneto-hydrodynamics and reference magnetic coordinates for simulations of plasma disruptions

    SciTech Connect

    Zakharov, Leonid E.; Li, Xujing

    2015-06-15

    This paper formulates the Tokamak Magneto-Hydrodynamics (TMHD), initially outlined by X. Li and L. E. Zakharov [Plasma Science and Technology 17(2), 97–104 (2015)] for proper simulations of macroscopic plasma dynamics. The simplest set of magneto-hydrodynamics equations, sufficient for disruption modeling and extendable to more refined physics, is explained in detail. First, the TMHD introduces to 3-D simulations the Reference Magnetic Coordinates (RMC), which are aligned with the magnetic field in the best possible way. The numerical implementation of RMC is adaptive grids. Being consistent with the high anisotropy of the tokamak plasma, RMC allow simulations at realistic, very high plasma electric conductivity. Second, the TMHD splits the equation of motion into an equilibrium equation and the plasma advancing equation. This resolves the 4 decade old problem of Courant limitations of the time step in existing, plasma inertia driven numerical codes. The splitting allows disruption simulations on a relatively slow time scale in comparison with the fast time of ideal MHD instabilities. A new, efficient numerical scheme is proposed for TMHD.

  3. Temperature measurement of a dust particle in a RF plasma GEC reference cell

    NASA Astrophysics Data System (ADS)

    Kong, Jie; Qiao, Ke; Matthews, Lorin S.; Hyde, Truell W.

    2016-10-01

    The thermal motion of a dust particle levitated in a plasma chamber is similar to that described by Brownian motion in many ways. The primary difference between a dust particle in a plasma system and a free Brownian particle is that in addition to the random collisions between the dust particle and the neutral gas atoms, there are electric field fluctuations, dust charge fluctuations, and correlated motions from the unwanted continuous signals originating within the plasma system itself. This last contribution does not include random motion and is therefore separable from the random motion in a `normal' temperature measurement. In this paper, we discuss how to separate random and coherent motions of a dust particle confined in a glass box in a Gaseous Electronic Conference (GEC) radio-frequency (RF) reference cell employing experimentally determined dust particle fluctuation data analysed using the mean square displacement technique.

  4. Detection, identification, and quantification of selenoproteins in a candidate human plasma standard reference material.

    PubMed

    Ballihaut, Guillaume; Kilpatrick, Lisa E; Davis, W Clay

    2011-11-15

    To understand the effect of Se supplementation on health, it is critical to accurately assess the Se status in the human body by measuring reliable biomarkers. The preferred biomarkers of the Se status are selenoprotein P (SelP) and glutathione peroxidase 3 (GPx3) along with selenoalbumin (SeAlb), but there is still a real need for reference methods and reference materials to validate their measurements. Therefore, this work presents a systematic approach to provide quality control data in selenoprotein measurements. This approach combines online isotope dilution affinity liquid chromatography (LC) coupled to inductively coupled plasma mass spectrometry (ICPMS), laser ablation ICPMS, and tandem mass spectrometry (MS/MS) to identify and quantify SelP, GPx3, and SeAlb in a human plasma reference material SRM 1950. Quantitative determinations of SelP, GPx3, and SeAlb were 50.2 ± 4.3, 23.6 ± 1.3, and 28.2 ± 2.6 ng g(-1) as Se, respectively. The subsequent identification of the selenoproteins included nine SelP peptides, including two selenopeptides and nine GPx3 peptides, while albumin was identified with a protein coverage factor >95%. The structural elucidation of selenoproteins in the target Se affinity fractions in SRM 1950 provides information needed for method validation and quality control measurements of selenoproteins and therefore the selenium status in human plasma.

  5. Plasma Proteomic Profiling in Hereditary Breast Cancer Reveals a BRCA1-Specific Signature: Diagnostic and Functional Implications.

    PubMed

    Scumaci, Domenica; Tammè, Laura; Fiumara, Claudia Vincenza; Pappaianni, Giusi; Concolino, Antonio; Leone, Emanuela; Faniello, Maria Concetta; Quaresima, Barbara; Ricevuto, Enrico; Costanzo, Francesco Saverio; Cuda, Giovanni

    2015-01-01

    Breast cancer (BC) is a leading cause of death among women. Among the major risk factors, an important role is played by familial history of BC. Germ-line mutations in BRCA1/2 genes account for most of the hereditary breast and/or ovarian cancers. Gene expression profiling studies have disclosed specific molecular signatures for BRCA1/2-related breast tumors as compared to sporadic cases, which might help diagnosis and clinical follow-up. Even though, a clear hallmark of BRCA1/2-positive BC is still lacking. Many diseases are correlated with quantitative changes of proteins in body fluids. Plasma potentially carries important information whose knowledge could help to improve early disease detection, prognosis, and response to therapeutic treatments. The aim of this study was to develop a comprehensive approach finalized to improve the recovery of specific biomarkers from plasma samples of subjects affected by hereditary BC. To perform this analysis, we used samples from patients belonging to highly homogeneous population previously reported. Depletion of high abundant plasma proteins, 2D gel analysis, liquid chromatography-tandem mass spectrometry (LC-MS/MS) and bioinformatics analysis were used into an integrated approach to investigate tumor-specific changes in the plasma proteome of BC patients and healthy family members sharing the same BRCA1 gene founder mutation (5083del19), previously reported by our group, with the aim to identify specific signatures. The comparative analysis of the experimental results led to the identification of gelsolin as the most promising biomarker. Further analyses, performed using a panel of breast cancer cell lines, allowed us to further elucidate the signaling network that might modulate the expression of gelsolin in breast cancer.

  6. Plasma Membrane Proteomics of Human Breast Cancer Cell Lines Identifies Potential Targets for Breast Cancer Diagnosis and Treatment

    PubMed Central

    Ziegler, Yvonne S.; Moresco, James J.; Tu, Patricia G.; Yates, John R.; Nardulli, Ann M.

    2014-01-01

    The use of broad spectrum chemotherapeutic agents to treat breast cancer results in substantial and debilitating side effects, necessitating the development of targeted therapies to limit tumor proliferation and prevent metastasis. In recent years, the list of approved targeted therapies has expanded, and it includes both monoclonal antibodies and small molecule inhibitors that interfere with key proteins involved in the uncontrolled growth and migration of cancer cells. The targeting of plasma membrane proteins has been most successful to date, and this is reflected in the large representation of these proteins as targets of newer therapies. In view of these facts, experiments were designed to investigate the plasma membrane proteome of a variety of human breast cancer cell lines representing hormone-responsive, ErbB2 over-expressing and triple negative cell types, as well as a benign control. Plasma membranes were isolated by using an aqueous two-phase system, and the resulting proteins were subjected to mass spectrometry analysis. Overall, each of the cell lines expressed some unique proteins, and a number of proteins were expressed in multiple cell lines, but in patterns that did not always follow traditional clinical definitions of breast cancer type. From our data, it can be deduced that most cancer cells possess multiple strategies to promote uncontrolled growth, reflected in aberrant expression of tyrosine kinases, cellular adhesion molecules, and structural proteins. Our data set provides a very rich and complex picture of plasma membrane proteins present on breast cancer cells, and the sorting and categorizing of this data provides interesting insights into the biology, classification, and potential treatment of this prevalent and debilitating disease. PMID:25029196

  7. Differential proteomic analysis of virus-enriched fractions obtained from plasma pools of patients with dengue fever or severe dengue.

    PubMed

    Fragnoud, Romain; Flamand, Marie; Reynier, Frederic; Buchy, Philippe; Duong, Vasna; Pachot, Alexandre; Paranhos-Baccala, Glaucia; Bedin, Frederic

    2015-11-14

    Dengue is the most widespread mosquito-borne viral disease of public health concern. In some patients, endothelial cell and platelet dysfunction lead to life-threatening hemorrhagic dengue fever or dengue shock syndrome. Prognostication of disease severity is urgently required to improve patient management. The pathogenesis of severe dengue has not been fully elucidated, and the role of host proteins associated with viral particles has received little exploration. The proteomes of virion-enriched fractions purified from plasma pools of patients with dengue fever or severe dengue were compared. Virions were purified by ultracentrifugation combined with a water-insoluble polyelectrolyte-based technique. Following in-gel hydrolysis, peptides were analyzed by nano-liquid chromatography coupled to ion trap mass spectrometry and identified using data libraries. Both dengue fever and severe dengue viral-enriched fractions contained identifiable viral envelope proteins and host cellular proteins. Canonical pathway analysis revealed the identified host proteins are mainly involved in the coagulation cascade, complement pathway or acute phase response signaling pathway. Some host proteins were over- or under-represented in plasma from patients with severe dengue compared to patients with dengue fever. ELISAs were used to validate differential expression of a selection of identified host proteins in individual plasma samples of patients with dengue fever compared to patients with severe dengue. Among 22 host proteins tested, two could differentiate between dengue fever and severe dengue in two independent cohorts (olfactomedin-4: area under the curve (AUC), 0.958; and platelet factor-4: AUC, 0.836). A novel technique of virion-enrichment from plasma has allowed to identify two host proteins that have prognostic value for classifying patients with acute dengue who are more likely to develop a severe dengue. The impact of these host proteins on pathogenicity and disease outcome

  8. Comparative analysis of boar seminal plasma proteome from different freezability ejaculates and identification of Fibronectin 1 as sperm freezability marker.

    PubMed

    Vilagran, I; Yeste, M; Sancho, S; Castillo, J; Oliva, R; Bonet, S

    2015-03-01

    Variation in boar sperm freezability (i.e. capacity to withstand cryopreservation) between ejaculates is a limitation largely reported in the literature. Prediction of sperm freezability and classification of boar ejaculates into good (GFEs) and poor freezability ejaculates (PFEs) before cryopreservation takes place may increase the use of frozen-thawed spermatozoa. While markers of boar sperm freezability have been found from sperm cell extracts, little attention has been paid to seminal plasma. On this basis, the present study compared the fresh seminal plasma proteome of 9 GFEs and 9 PFEs through two-dimensional difference gel electrophoresis (2D-DIGE) and liquid chromatography mass spectrometry (LC-MS/MS). The ejaculates were previously classified as GFE or PFE upon their sperm viability and progressive motility assessments at 30 and 240 min post thawing. From a total of 51 spots, four were found to significantly (p < 0.05) differ between GFEs and PFEs, and two were identified as fibronectin-1 (FN1) and glutathione peroxidase 5 (GPX5). These two potential markers were further studied by western blot and correlation analysis between protein relative abundances in fresh seminal plasma and regression factors from principal component analyses (PCA) run using post-thawing sperm quality parameters. Results confirmed that FN1 is a reliable marker of boar sperm freezability, because GFEs presented significantly (p < 0.05) higher FN1-amounts than PFEs and FN1 was found to be correlated with the first PCA component at 240 min post thawing. In contrast, GPX5 was not validated as a boar sperm freezability marker. We can thus conclude that levels of FN1 in fresh seminal plasma from boar semen may be used as a sperm freezability marker, thereby facilitating the use of frozen-thawed boar spermatozoa.

  9. Derivation of reference distribution functions for Tokamak-plasmas by statistical thermodynamics

    NASA Astrophysics Data System (ADS)

    Sonnino, Giorgio; Cardinali, Alessandro; Peeters, Philippe; Steinbrecher, György; Sonnino, Alberto; Nardone, Pasquale

    2014-03-01

    A general approach for deriving the expression of reference distribution functions by statistical thermodynamics is illustrated, and applied to the case of a magnetically confined plasma. The local equilibrium is defined by imposing the minimum entropy production, which applies only to the linear regime near a stationary thermodynamically non-equilibrium state and the maximum entropy principle under the scale invariance restrictions. This procedure may be adopted for a system subject to an arbitrary number of thermodynamic forces, however, for concreteness, we analyze, afterwords, a magnetically confined plasma subject to three thermodynamic forces, and three energy sources: (i) the total Ohmic heat, supplied by the transformer coil; (ii) the energy supplied by neutral beam injection (NBI); and (iii) the RF energy supplied by ion cyclotron resonant heating (ICRH) system which heats the minority population. In this limit case, we show that the derived expression of the distribution function is more general than that one, which is currently used for fitting the numerical steady-state solutions obtained by simulating the plasma by gyro-kinetic codes. An application to a simple model of fully ionized plasmas submitted to an external source is discussed. Through kinetic theory, we fixed the values of the free parameters linking them with the external power supplies. The singularity at low energy in the proposed distribution function is related to the intermittency in the turbulent plasma.

  10. Connecting genetic risk to disease end points through the human blood plasma proteome

    PubMed Central

    Suhre, Karsten; Arnold, Matthias; Bhagwat, Aditya Mukund; Cotton, Richard J.; Engelke, Rudolf; Raffler, Johannes; Sarwath, Hina; Thareja, Gaurav; Wahl, Annika; DeLisle, Robert Kirk; Gold, Larry; Pezer, Marija; Lauc, Gordan; El-Din Selim, Mohammed A.; Mook-Kanamori, Dennis O.; Al-Dous, Eman K.; Mohamoud, Yasmin A.; Malek, Joel; Strauch, Konstantin; Grallert, Harald; Peters, Annette; Kastenmüller, Gabi; Gieger, Christian; Graumann, Johannes

    2017-01-01

    Genome-wide association studies (GWAS) with intermediate phenotypes, like changes in metabolite and protein levels, provide functional evidence to map disease associations and translate them into clinical applications. However, although hundreds of genetic variants have been associated with complex disorders, the underlying molecular pathways often remain elusive. Associations with intermediate traits are key in establishing functional links between GWAS-identified risk-variants and disease end points. Here we describe a GWAS using a highly multiplexed aptamer-based affinity proteomics platform. We quantify 539 associations between protein levels and gene variants (pQTLs) in a German cohort and replicate over half of them in an Arab and Asian cohort. Fifty-five of the replicated pQTLs are located in trans. Our associations overlap with 57 genetic risk loci for 42 unique disease end points. We integrate this information into a genome-proteome network and provide an interactive web-tool for interrogations. Our results provide a basis for novel approaches to pharmaceutical and diagnostic applications. PMID:28240269

  11. Assessment of suitability of magnetic beads for purification of rat plasma in proteomic analyses by matrix-assisted laser desorption ionization-time-of-flight MS.

    PubMed

    Mohottalage, Susantha; Vincent, Renaud; Kumarathasan, Prem

    2009-01-01

    Plasma is a complex matrix and has to be clarified or fractionated to obtain informative MS data. Although there are a number of prefractionation methods to clean up complex biological matrixes before proteomic analysis, these methods require large sample volumes and are costly and time-consuming. Alternatively, recently introduced magnetic beads (MB) appear to be attractive in overcoming these difficulties. Therefore, we were interested in investigating the applicability of MB in the clarification of rat plasma samples for proteome analyses. For this purpose, we used complementary supports, such as hydrophobic interaction chromatography-based MB (MB-C18) and weak cation-exchange chromatography-based MB (MB-WCX). MB-based fractionated samples were either spotted directly or underwent tryptic digestion before matrix-assisted laser desorption ionization (MALDI) spotting. Samples from both MB separation techniques gave clean and well-resolved MALDI-time-of-flight MS spectra in the low molecular mass range of 1-10 kDa with alpha-cyano-4-hydroxycinnamic acid as the matrix. Both techniques gave approximately 300 analyte peaks in this mass range. Our results showed that both MB-based separation procedures gave complementary mass spectral information. This approach provided information on the identity of a number of less-abundant and more-abundant proteins in plasma. Our findings suggest that this MB-based proteomic approach can be valuable in conducting faster screening of plasma samples for protein profiling.

  12. Enhanced dietary formulation to mitigate winter thermal stress in gilthead sea bream (Sparus aurata): a 2D-DIGE plasma proteome study.

    PubMed

    Schrama, Denise; Richard, Nadège; Silva, Tomé S; Figueiredo, Filipe A; Conceição, Luís E C; Burchmore, Richard; Eckersall, David; Rodrigues, Pedro M L

    2016-11-24

    Low water temperatures during winter are common in farming of gilthead sea bream in the Mediterranean. This causes metabolic disorders that in extreme cases can lead to a syndrome called "winter disease." An improved immunostimulatory nutritional status might mitigate the effects of this thermal metabolic stress. A trial was set up to assess the effects of two different diets on gilthead sea bream physiology and nutritional state through plasma proteome and metabolites. Four groups of 25 adult gilthead sea bream were reared during winter months, being fed either with a control diet (CTRL) or with a diet called "winter feed" (WF). Proteome results show a slightly higher number of proteins upregulated in plasma of fish fed the WF. These proteins are mostly involved in the immune system and cell protection mechanisms. Lipid metabolism was also affected, as shown both by plasma proteome and by the cholesterol plasma levels. Overall, the winter feed diet tested seems to have positive effects in terms of fish condition and nutritional status, reducing the metabolic effects of thermal stress.

  13. Reference change values of plasma and urine NGAL in cardiac surgery with cardiopulmonary bypass.

    PubMed

    Bataille, Aurélien; Tiepolo, Ambre; Robert, Tiphaine; Boutten, Anne; Longrois, Dan; Dehoux, Monique; Provenchère, Sophie

    2017-09-23

    As with any biomarker, interpretation of changes of NGAL concentration must consider its variability in a specific clinical setting. The aim of this study was to calculate the reference change value (RCV) and the index of individuality (II) of plasma and urine NGAL in the context of coronary artery bypass graft surgery with cardiopulmonary bypass, in patients without postoperative acute kidney injury. This prospective single-center observational study included patients with a preoperative glomerular filtration rate of >30mlmin(-1) 1.73m(-2), scheduled for elective coronary artery bypass graft with cardiopulmonary bypass and free from postoperative renal injury according to KDIGO criteria during hospital stay or a plasma creatinine Δ<0 (Δ=day1-induction). Plasma and urine NGAL were measured at anesthesia induction, 4h after intensive care admission and on the first and 2nd postoperative day and normalized to plasma proteins or urine creatinine. The RCV was given by the formula: 1.96×√2×√(CVa(2)+CVi(2)), were CVi is the intra-individual variability and CVa the reported analytical coefficient of variation of 5%. The II was calculated using the formula II=CVi/CVg for the four previous parameters, where CVg is the inter-individual variability. Of the 100 patients enrolled in the study, 73 or 25 were considered free from acute kidney injury (KDIGO and Δ creatinine criteria, respectively) and included in the analysis. The RCV was 104% and 109% for plasma NGAL and 321% and 608% for urine NGAL. The II was <0.6 for both plasma and urine NGAL. In patients who underwent coronary artery bypass grafting with normal post-operative kidney function, two-fold change in plasma NGAL and three to six-fold change in urine NGAL occur. In this specific clinical context, pathological variations must consider this biological "noise" for correct interpretation. Copyright © 2017. Published by Elsevier Inc.

  14. Analysis of biological reference materials, prepared by microwave dissolution, using inductively coupled plasma mass spectrometry.

    PubMed

    Friel, J K; Skinner, C S; Jackson, S E; Longerich, H P

    1990-03-01

    A procedure has been developed for the analysis of biological materials by inductively coupled plasma mass spectrometry (ICP-MS). Fast, efficient and complete sample digestion is achieved by a combined microwave-nitric acid/open beaker-nitric acid-hydrogen peroxide procedure. The ICP-MS analysis is performed with an on-line five-element internal standard to correct for matrix and instrumental drift effects. Results are presented for 24 elements in three biological reference materials (National Institute of Standards and Technology Standard Reference Materials 5277a Liver and 1566 Oyster and International Atomic Energy Agency Certified Reference Material H4 Animal Muscle). For all elements significantly above the detection limit and reagent blank concentrations, good agreement exists between ICP-MS and certified values.

  15. Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma.

    PubMed

    Kalra, Hina; Adda, Christopher G; Liem, Michael; Ang, Ching-Seng; Mechler, Adam; Simpson, Richard J; Hulett, Mark D; Mathivanan, Suresh

    2013-11-01

    Exosomes are nanovesicles released by a variety of cells and are detected in body fluids including blood. Recent studies have highlighted the critical application of exosomes as personalized targeted drug delivery vehicles and as reservoirs of disease biomarkers. While these research applications have created significant interest and can be translated into practice, the stability of exosomes needs to be assessed and exosome isolation protocols from blood plasma need to be optimized. To optimize methods to isolate exosomes from blood plasma, we performed a comparative evaluation of three exosome isolation techniques (differential centrifugation coupled with ultracentrifugation, epithelial cell adhesion molecule immunoaffinity pull-down, and OptiPrep(TM) density gradient separation) using normal human plasma. Based on MS, Western blotting and microscopy results, we found that the OptiPrep(TM) density gradient method was superior in isolating pure exosomal populations, devoid of highly abundant plasma proteins. In addition, we assessed the stability of exosomes in plasma over 90 days under various storage conditions. Western blotting analysis using the exosomal marker, TSG101, revealed that exosomes are stable for 90 days. Interestingly, in the context of cellular uptake, the isolated exosomes were able to fuse with target cells revealing that they were indeed biologically active.

  16. Plasma membrane proteomes of differentially matured dendritic cells identified by LC–MS/MS combined with iTRAQ labelling

    PubMed Central

    Ferret-Bernard, Stéphanie; Castro-Borges, William; Dowle, Adam A.; Sanin, David E.; Cook, Peter C.; Turner, Joseph D.; MacDonald, Andrew S.; Thomas, Jerry R.; Mountford, Adrian P.

    2012-01-01

    Dendritic cells (DCs) play a pivotal role in polarising Th lymphocyte subsets but it is unclear what molecular events occur when DCs generate Th2-type responses. Here, we analysed plasma membrane-enriched fractions from immature, pro-Th1 and pro-Th2 DCs and used a combination of iTRAQ labelling and LC–MS/MS to quantify changes in the proteomes. Analysis was performed on triplicate biological samples and changes verified by flow cytometry. MHC class II molecules and CD29 were up-regulated in pro-Th1 DCs whilst CD18 and CD44 were up-regulated in pro-Th2 DCs. One of the most down-regulated molecules in pro-Th1 DCs was YM-1 whilst the greatest decrease in pro-Th2 DCs was NAP-22. Other molecules up-regulated in pro-Th2 DC compared to pro-Th1 DCs included some potentially involved in protein folding during antigen processing (clathrin and Rab-7), whilst other non-membrane proteins such as enzymes/transporters related to cell metabolism (malate dehydrogenase, pyruvate kinase, and ATPase Na+/K+) were also recorded. This suggests that pro-Th2 DCs are more metabolically active while pro-Th1 DCs have a mature ‘end state’. Overall, although several molecules were preferentially expressed on pro-Th2 DCs, our proteomics data support the view of a ‘limited maturation’ of pro-Th2 DCs compared to pro-Th1 DCs. PMID:22040742

  17. Randomized Trial of Glucosamine and Chondroitin Supplementation on Inflammation and Oxidative Stress Biomarkers and Plasma Proteomics Profiles in Healthy Humans

    PubMed Central

    Navarro, Sandi L.; White, Emily; Kantor, Elizabeth D.; Zhang, Yuzheng; Rho, Junghyun; Song, Xiaoling; Milne, Ginger L.; Lampe, Paul D.; Lampe, Johanna W.

    2015-01-01

    Background Glucosamine and chondroitin are popular non-vitamin dietary supplements used for osteoarthritis. Long-term use is associated with lower incidence of colorectal and lung cancers and with lower mortality; however, the mechanism underlying these observations is unknown. In vitro and animal studies show that glucosamine and chondroitin inhibit NF-kB, a central mediator of inflammation, but no definitive trials have been done in healthy humans. Methods We conducted a randomized, double-blind, placebo-controlled, cross-over study to assess the effects of glucosamine hydrochloride (1500 mg/d) plus chondroitin sulfate (1200 mg/d) for 28 days compared to placebo in 18 (9 men, 9 women) healthy, overweight (body mass index 25.0–32.5 kg/m2) adults, aged 20–55 y. We examined 4 serum inflammatory biomarkers: C-reactive protein (CRP), interleukin 6, and soluble tumor necrosis factor receptors I and II; a urinary inflammation biomarker: prostaglandin E2-metabolite; and a urinary oxidative stress biomarker: F2-isoprostane. Plasma proteomics on an antibody array was performed to explore other pathways modulated by glucosamine and chondroitin. Results Serum CRP concentrations were 23% lower after glucosamine and chondroitin compared to placebo (P = 0.048). There were no significant differences in other biomarkers. In the proteomics analyses, several pathways were significantly different between the interventions after Bonferroni correction, the most significant being a reduction in the “cytokine activity” pathway (P = 2.6 x 10-16), after glucosamine and chondroitin compared to placebo. Conclusion Glucosamine and chondroitin supplementation may lower systemic inflammation and alter other pathways in healthy, overweight individuals. This study adds evidence for potential mechanisms supporting epidemiologic findings that glucosamine and chondroitin are associated with reduced risk of lung and colorectal cancer. Trial Registration ClinicalTrials.gov NCT01682694 PMID

  18. Overview of the HUPO Plasma Proteome Project: Results from the pilot phase with 35 collaborating laboratories and multiple analytical groups, generating a core dataset of 3020 proteins and a publicly-available database

    SciTech Connect

    Omenn, Gilbert; States, David J.; Adamski, Marcin; Blackwell, Thomas W.; Menon, Rajasree; Hermjakob, Henning; Apweiler, Rolf; Haab, Brian B.; Simpson, Richard; Eddes, James; Kapp, Eugene; Moritz, Rod; Chan, Daniel W.; Rai, Alex J.; Admon, Arie; Aebersold, Ruedi; Eng, Jimmy K.; Hancock, William S.; Hefta, Stanley A.; Meyer, Helmut; Paik, Young-Ki; Yoo, Jong-Shin; Ping, Peipei; Pounds, Joel G.; Adkins, Joshua N.; Qian, Xiaohong; Wang, Rong; Wasinger, Valerie; Wu, Chi Yue; Zhao, Xiaohang; Zeng, Rong; Archakov, Alexander; Tsugita, Akira; Beer, Ilan; Pandey, Akhilesh; Pisano, Michael; Andrews, Philip; Tammen, Harald; Speicher, David W.; Hanash, Samir M.

    2005-08-13

    HUPO initiated the Plasma Proteome Project (PPP) in 2002. Its pilot phase has (1) evaluated advantages and limitations of many depletion, fractionation, and MS technology platforms; (2) compared PPP reference specimens of human serum and EDTA, heparin, and citrate-anticoagulated plasma; and (3) created a publicly-available knowledge base (www.bioinformatics. med.umich.edu/hupo/ppp; www.ebi.ac.uk/pride). Thirty-five participating laboratories in 13 countries submitted datasets. Working groups addressed (a) specimen stability and protein concentrations; (b) protein identifications from 18 MS/MS datasets; (c) independent analyses from raw MS-MS spectra; (d) search engine performance, subproteome analyses, and biological insights; (e) antibody arrays; and (f) direct MS/SELDI analyses. MS-MS datasets had 15 710 different International Protein Index (IPI) protein IDs; our integration algorithm applied to multiple matches of peptide sequences yielded 9504 IPI proteins identified with one or more peptides and 3020 proteins identified with two or more peptides (the Core Dataset). These proteins have been characterized with Gene Ontology, InterPro, Novartis Atlas, OMIM, and immunoassay based concentration determinations. The database permits examination of many other subsets, such as 1274 proteins identified with three or more peptides. Reverse protein to DNA matching identified proteins for 118 previously unidentified ORFs. We recommend use of plasma instead of serum, with EDTA (or citrate) for anticoagulation. To improve resolution, sensitivity and reproducibility of peptide identifications and protein matches, we recommend combinations of depletion, fractionation, and MS/MS technologies, with explicit criteria for evaluation of spectra, use of search algorithms, and integration of homologous protein matches. This Special Issue of PROTEOMICS presents papers integral to the collaborative analysis plus many reports of supplementary work on various aspects of the PPP workplan

  19. A reference growth curve for nutritional experiments in zebrafish (Danio rerio) and changes in whole body proteome during development.

    PubMed

    Gómez-Requeni, P; Conceição, L E C; Olderbakk Jordal, A-E; Rønnestad, I

    2010-12-01

    Zebrafish is one of the most used vertebrate model organisms in molecular and developmental biology, recently gaining popularity also in medical research. However, very little work has been done to assess zebrafish as a model species in nutritional studies in aquaculture in order to utilize the methodological toolbox that this species represents. As a starting point to acquire some baseline data for further nutritional studies, growth of a population of zebrafish was followed for 15 weeks. Furthermore, whole body proteome was screened during development by means of bi-dimensional gel electrophoresis and mass spectrometry. Fish were reared under best practice laboratory conditions from hatching until 103 days post-fertilization (dpf) and regularly fed ad libitum with Artemia nauplii from 12 dpf. A growth burst occurred within 9-51 dpf, reaching a plateau after 65 dpf. Fork length and body weight were significantly lower in males than in females from 58 dpf onwards. Proteomics analysis showed 28 spot proteins differently expressed through development and according to sex. Of these proteins, 20 were successfully identified revealing proteins involved in energy production, muscle development, eye lens differentiation, and sexual maturation. In summary, zebrafish exhibited a rapid growth until approximately 50 dpf, when most individuals started to allocate part of the dietary energy intake for sexual maturation. However, proteomic analysis revealed that some individuals reached sexual maturity earlier and already from 30 dpf onwards. Thus, in order to design nutritional studies with zebrafish fed Artemia nauplii, it is recommended to select a period between 20 and 40 dpf, when fish allocate most of the ingested energy for non-reproductive growth purposes.

  20. Multicentric study of the effect of pre-analytical variables in the quality of plasma samples stored in biobanks using different complementary proteomic methods.

    PubMed

    Mateos, Jesús; Carneiro, Isabel; Corrales, Fernando; Elortza, Felix; Paradela, Alberto; Del Pino, Manuel Sánchez; Iloro, Ibon; Marcilla, Miguel; Mora, Maria Isabel; Valero, Luz; Ciordia, Sergio; Fernández, Verónica; Fortuño, Maria Antonia; García-Sánchez, Isabel; Martínez, Rosario; Muñoz, Maria Angeles; Rodriguez, Clara; Doménech, Nieves

    2017-01-06

    Analytical proteomics has experienced exponential progress in the last decade and can be expected to lead research studies on diagnostic and therapeutic biomarkers in the near future. Because the development of this type of analysis requires the use of a large number of human samples with a minimum of quality requirements, our objective was to identify appropriate indicators for quality control of plasma samples stored in biobanks for research in proteomics. To accomplish this, plasma samples from 100 healthy donors were obtained and processed according to the pre-analytical variables of: a) time delay for the first centrifugation of the original blood sample (4 or 24h) and b) number of freeze/thaw cycles (1, 2 or 3) of the processed plasma samples. The analyses of samples were performed by different and complementary methods such as SPE MALDI-TOF, DIGE, shotgun (iTRAQ, nLC MALDI TOF/TOF) and targeted nLC MS/MS proteomic techniques (SRM). In general, because the distribution of proteins in all samples was found to be very similar, the results shown that delayed processing of blood samples and the number of freeze/thaw cycles has little or no effect on the integrity of proteins in the plasma samples. The results of the present work indicate that blood proteins in plasma are broadly insensitive to such preanalytical variables as delayed processing or freeze/thaw cycles when analyzed at the peptide level. Although there are other studies related to protein stability of clinical samples with similar results, what is remarkable about our work is the large number of plasma samples examined and that our analyses assessed protein integrity by combining a wide set of complementary proteomic approaches performed at different proteomic platform participating laboratories that all yielded similar results. We believe our study is the most comprehensive performed to date to determine the changes in proteins induced by delayed sample processing and plasma freeze/thaw cycles

  1. Validity of extracellular water assessment with saliva samples using plasma as the reference biological fluid.

    PubMed

    Matias, Catarina N; Silva, Analiza M; Santos, Diana A; Gobbo, Luis A; Schoeller, Dale A; Sardinha, Luís B

    2012-11-01

    Extracellular water (ECW) assessment is based on dilution techniques, commonly using blood sampling. However, plasma collection is an invasive procedure. We aimed to validate the use of saliva for ECW estimation by the bromide dilution technique using plasma as the reference method, in a sample of elite athletes. A total of 89 elite athletes with a mean age of 20.4 ± 4.4 years were evaluated. Baseline samples were collected before sodium bromide oral dose administration, and enriched samples were collected 3 h post-dose administration. The bromide concentration was assessed by high-performance liquid chromatography. Comparison of means, concordance coefficient correlation (CCC), multiple regression and Bland-Altman analysis were performed. The ECW from saliva explained 91% of the variance in ECW by plasma with a standard error of estimation of 0.91 kg. The CCC between alternative and reference methods was 0.952. No significant trend was observed between the mean and difference of the methods, with limits of agreement ranging between -1.5 and 2.1 kg. These findings reveal that bromide dilution volume calculated from saliva samples is a valid noninvasive method for ECW assessment in elite athletes.

  2. Identification of plasma biomarker candidates in glioblastoma using an antibody-array-based proteomic approach.

    PubMed

    Zupancic, Klemen; Blejec, Andrej; Herman, Ana; Veber, Matija; Verbovsek, Urska; Korsic, Marjan; Knezevic, Miomir; Rozman, Primoz; Turnsek, Tamara Lah; Gruden, Kristina; Motaln, Helena

    2014-09-01

    Glioblastoma multiforme (GBM) is a brain tumour with a very high patient mortality rate, with a median survival of 47 weeks. This might be improved by the identification of novel diagnostic, prognostic and predictive therapy-response biomarkers, preferentially through the monitoring of the patient blood. The aim of this study was to define the impact of GBM in terms of alterations of the plasma protein levels in these patients. We used a commercially available antibody array that includes 656 antibodies to analyse blood plasma samples from 17 healthy volunteers in comparison with 17 blood plasma samples from patients with GBM. We identified 11 plasma proteins that are statistically most strongly associated with the presence of GBM. These proteins belong to three functional signalling pathways: T-cell signalling and immune responses; cell adhesion and migration; and cell-cycle control and apoptosis. Thus, we can consider this identified set of proteins as potential diagnostic biomarker candidates for GBM. In addition, a set of 16 plasma proteins were significantly associated with the overall survival of these patients with GBM. Guanine nucleotide binding protein alpha (GNAO1) was associated with both GBM presence and survival of patients with GBM. Antibody array analysis represents a useful tool for the screening of plasma samples for potential cancer biomarker candidates in small-scale exploratory experiments; however, clinical validation of these candidates requires their further evaluation in a larger study on an independent cohort of patients.

  3. Identification of plasma biomarker candidates in glioblastoma using an antibody-array-based proteomic approach

    PubMed Central

    Zupancic, Klemen; Blejec, Andrej; Herman, Ana; Veber, Matija; Verbovsek, Urska; Korsic, Marjan; Knezevic, Miomir; Rozman, Primoz; Turnsek, Tamara Lah; Gruden, Kristina; Motaln, Helena

    2014-01-01

    Background Glioblastoma multiforme (GBM) is a brain tumour with a very high patient mortality rate, with a median survival of 47 weeks. This might be improved by the identification of novel diagnostic, prognostic and predictive therapy-response biomarkers, preferentially through the monitoring of the patient blood. The aim of this study was to define the impact of GBM in terms of alterations of the plasma protein levels in these patients. Materials and methods. We used a commercially available antibody array that includes 656 antibodies to analyse blood plasma samples from 17 healthy volunteers in comparison with 17 blood plasma samples from patients with GBM. Results We identified 11 plasma proteins that are statistically most strongly associated with the presence of GBM. These proteins belong to three functional signalling pathways: T-cell signalling and immune responses; cell adhesion and migration; and cell-cycle control and apoptosis. Thus, we can consider this identified set of proteins as potential diagnostic biomarker candidates for GBM. In addition, a set of 16 plasma proteins were significantly associated with the overall survival of these patients with GBM. Guanine nucleotide binding protein alpha (GNAO1) was associated with both GBM presence and survival of patients with GBM. Conclusions Antibody array analysis represents a useful tool for the screening of plasma samples for potential cancer biomarker candidates in small-scale exploratory experiments; however, clinical validation of these candidates requires their further evaluation in a larger study on an independent cohort of patients. PMID:25177240

  4. Constructing Proteome Reference Map of the Porcine Jejunal Cell Line (IPEC-J2) by Label-Free Mass Spectrometry.

    PubMed

    Kim, Sang Hoon; Pajarillo, Edward Alain B; Balolong, Marilen P; Lee, Ji Yoon; Kang, Dae-Kyung

    2016-06-28

    In this study, the global proteome of the IPEC-J2 cell line was evaluated using ultra-high performance liquid chromatography coupled to a quadrupole Q Exactive™ Orbitrap mass spectrometer. Proteins were isolated from highly confluent IPEC-J2 cells in biological replicates and analyzed by label-free mass spectrometry prior to matching against a porcine genomic dataset. The results identified 1,517 proteins, accounting for 7.35% of all genes in the porcine genome. The highly abundant proteins detected, such as actin, annexin A2, and AHNAK nucleoprotein, are involved in structural integrity, signaling mechanisms, and cellular homeostasis. The high abundance of heat shock proteins indicated their significance in cellular defenses, barrier function, and gut homeostasis. Pathway analysis and annotation using the Kyoto Encyclopedia of Genes and Genomes database resulted in a putative protein network map of the regulation of immunological responses and structural integrity in the cell line. The comprehensive proteome analysis of IPEC-J2 cells provides fundamental insights into overall protein expression and pathway dynamics that might be useful in cell adhesion studies and immunological applications.

  5. Hematology and plasma biochemistry reference range values for free-ranging desert tortoises in Arizona.

    PubMed

    Dickinson, Vanessa M; Jarchow, James L; Trueblood, Mark H

    2002-01-01

    Baseline values and ranges for 10 hematologic and 32 plasma chemistry parameters were analyzed for 36 free-ranging Sonoran desert tortoises (Gopherus agassizzi) collected in Yavapai and La Paz Counties (Arizona, USA) from 1990 to 1995. Tortoises were radio tagged from 1990 to 1994, and attempts were made to recapture them three times a year. Tortoises were weighed, measured, and chemically immobilized to collect blood for hematology and blood chemistry assessments. Tortoise biochemistry differed (P < 0.01) between sites and sexes and among seasons and years. Normal reference ranges for hematologic and plasma biochemistry parameters were determined. Seasonal and annual differences in hematology and blood chemistry were related to rainfall patterns, forage availability, and physiological condition.

  6. Calculation of the surface tension of liquid metals using a one-component-plasma reference system

    NASA Technical Reports Server (NTRS)

    Zeng, X. C.; Stroud, D.

    1987-01-01

    The one-component-plasma (OCP) model is used as a reference system instead of the traditional hard-sphere fluid to calculate the liquid-vapor interfacial surface tension of liquid metals within the density functional formalism. The calculated surface tensions of the alkali metals are in excellent agreement with experiment. For the polyvalent metal Al, the result obtained is larger than experimental measurements. It is concluded that the OCP system is not suitable to describe the liquid-vapor phase transition in simple metals which have a nominal plasma parameter larger than the usual freezing value of about 178. The calculated interfacial widths in all cases are narrower than the expected experimental values.

  7. Oocyte proteomics: localisation of mouse zona pellucida protein 3 to the plasma membrane of ovulated mouse eggs.

    PubMed

    Coonrod, S A; Calvert, M E; Reddi, P P; Kasper, E N; Digilio, L C; Herr, J C

    2004-01-01

    In order to gain a deeper understanding of the molecular underpinnings of sperm-egg interaction and early development, we have used two-dimensional (2D) electrophoresis, avidin blotting and tandem mass spectrometry to identify, clone and characterise abundant molecules from the mouse egg proteome. Two-dimensional avidin blots of biotinylated zona-free eggs revealed an abundant approximately 75-kDa surface-labelled heterogeneous protein possessing a staining pattern similar to that of the zona pellucida glycoprotein, mouse ZP3 (mZP3). In light of this observation, we investigated whether mZP3 specifically localises to the plasma membrane of mature eggs. Zona pellucidae of immature mouse oocytes and mature eggs were removed using acid Tyrode's solution, chymotrypsin or mechanical shearing. Indirect immunofluorescence using the mZP3 monoclonal antibody (mAb) IE-10 demonstrated strong continuous staining over the entire surface of immature oocytes and weak microvillar staining on ovulated eggs, regardless of the method of zona removal. Interestingly, in mature eggs, increased fluorescence intensity was observed following artificial activation and fertilisation, whereas little to no fluorescence was observed in degenerated eggs. The surface localisation of ZP3 on mature eggs was supported by the finding that the IE-10 mAb immunoprecipitated an approximate 75-kDa protein from lysates of biotinylated zona-free eggs. To further investigate the specificity of the localisation of mZP3 to the oolemma, indirect immunofluorescence was performed using the IE-10 mAb on both CV-1 and CHO cells transfected with full-length recombinant mZP3 (re-mZP3). Plasma membrane targeting of the expressed re-mZP3 protein was observed in both cell lines. The membrane association of re-mZP3 was confirmed by the finding that biotinylated re-mZP3 (approximately 75 kDa) is immunoprecipitated from the hydrophobic phase of Triton X-114 extracts of transfected cells following phase partitioning

  8. Identification of Potential Plasma Biomarkers for Nonalcoholic Fatty Liver Disease by Integrating Transcriptomics and Proteomics in Laying Hens.

    PubMed

    Tsai, Meng-Tsz; Chen, Yu-Jen; Chen, Ching-Yi; Tsai, Mong-Hsun; Han, Chia-Li; Chen, Yu-Ju; Mersmann, Harry J; Ding, Shih-Torng

    2017-03-01

    Background: Prevalent worldwide obesity is associated with increased incidence of nonalcoholic fatty liver disease (NAFLD) and metabolic syndrome. The identification of noninvasive biomarkers for NAFLD is of recent interest. Because primary de novo lipogenesis occurs in chicken liver as in human liver, adult chickens with age-associated steatosis resembling human NAFLD is an appealing animal model.Objective: The objective of this study was to screen potential biomarkers in the chicken model for NAFLD by transcriptomic and proteomic analysis.Methods: Hy-Line W-36 laying hens were fed standard feed from 25 to 45 wk of age to induce fatty liver. They were killed every 4 wk, and liver and plasma were collected at each time point to assess fatty liver development and for transcriptomic and proteomic analysis. Next, selected biomarkers were confirmed in additional experiments by providing supplements of the hepatoprotective nutrients betaine [300, 600, or 900 parts per million (ppm) in vivo; 2 mM in vitro] or docosahexaenoic acid (DHA; 1% in vivo; 100 μM in vitro) to 30-wk-old Hy-Line W-36 laying hens for 4 mo and to Hy-Line W-36 chicken primary hepatocytes with oleic acid-induced steatosis. Liver or hepatocyte lipid contents and the expression of biomarkers were then examined.Results: Plasma acetoacetyl-CoA synthetase (AACS), dipeptidyl-peptidase 4 (DPP4), glutamine synthetase (GLUL), and glutathione S-transferase (GST) concentrations are well-established biomarkers for NAFLD. Selected biomarkers had significant positive associations with hepatic lipid deposition (P < 0.001). Betaine (900 ppm in vivo; 2 mM in vitro) and DHA (1% in vivo; 100 μM in vitro) supplementation both resulted in lower steatosis accompanied by the reduced expression of selected biomarkers in vivo and in vitro (P < 0.05).Conclusion: This study used adult laying hens to identify biomarkers for NAFLD and indicated that AACS, DPP4, GLUL, and GST could be considered to be potential diagnostic

  9. The plasma membrane proteome of Saccharomyces cerevisiae and its response to the antifungal calcofluor.

    PubMed

    Delom, Frédéric; Szponarski, Wojciech; Sommerer, Nicolas; Boyer, Jean-Christophe; Bruneau, Jean-Michel; Rossignol, Michel; Gibrat, Rémy

    2006-05-01

    Calcofluor is an antifungal compound known to induce structural perturbations of the cell wall by interfering with the synthesis of chitin microfibril. Proteins from a stripped plasma membrane fraction were solubilized with the neutral and non-denaturing detergent, the n-dodecyl beta-D-maltoside. Proteins were then resolved using a recently described ion-exchange chromatography (IEC)/lithium dodecyl sulfate (LDS)-PAGE procedure. Nearly 90 proteins were identified and clustered, based on their pI, molecular weight, abundance and/or hydrophobicity. This method was then applied to profile the plasma membrane response to calcofluor. The LDS-PAGE patterns obtained from whole plasma membrane proteins were similar for the non-treated and calcofluor-treated samples. However, IEC/LDS-PAGE analysis revealed subtle changes in the expression of several proteins of low abundance, in response to calcofluor. These proteins include Pil1p and Lsp1p, two sphingolipid long-chain base-responsive inhibitors of protein kinases involved in signaling pathways for cell wall integrity and Rho1p, a small GTPase. It was recently hypothesized that Pil1p and Lsp1p could associate with, and regulate, the plasma membrane beta-1-3-glucan synthase, responsible for the synthesis of another major microfibril for yeast cell wall. Results are discussed with respect to both calcofluor effects on the plasma membrane proteins and the power of the IEC/LDS-PAGE procedure in the search for new potential therapeutics targets.

  10. Determination of trace metals in marine biological reference materials by inductively coupled plasma mass spectrometry

    SciTech Connect

    Beauchemin, D.; McLaren, J.W.; Willie, S.N.; Berman, S.S.

    1988-04-01

    Inductively coupled plasma mass spectrometry (ICP-MS) was used for the analysis of two marine biological reference materials (dogfish liver tissue (DOLT-1) and dogfish muscle tissue (DORM-1)). The materials were put into solution by digestion in a nitric acid/hydrogen peroxide mixture. Thirteen elements (Na, Mg, Cr, Fe, Mn, Co, Ni, Cu, Zn, As, Cd, Hg, and Pb) were then determined. Accurate results were obtained by standard additions or isotope dilution techniques for all of these elements in DORM-1 and for all but Cr in DOLT-1.

  11. A proteomic approach to identify seminal plasma proteins in roosters (Gallus gallus domesticus).

    PubMed

    Marzoni, Margherita; Castillo, Annelisse; Sagona, Simona; Citti, Lorenzo; Rocchiccioli, Silvia; Romboli, Isabella; Felicioli, Antonio

    2013-08-01

    Considering the interest in avian semen processing and storage, the objective of this study was to identify the domestic fowl seminal plasma proteins using two-dimensional gel electrophoresis (2-DE) and mass spectrometry MS/MS. For three times in a 4-month period, seminal plasma was obtained from semen collected from four local male chickens (Gallus gallus domesticus) and prepared for two-dimensional polyacrylamide gel electrophoresis. A total of 83 spots were detected across all gels and analyzed by MALDI-TOF/TOF. Among these spots, 17 have been successfully identified. The most intensely stained spots were recognized as serum albumin, ovotransferrin, alpha-enolase, fatty acid binding protein, thioredoxin, trypsin inhibitor CITI-1 and gallinacin-9. From these proteins, two are characteristic of avian seminal plasma, the ovotransferrin and gallinacin-9, and one is specific of the Gallus species, the chicken trypsin inhibitor CITI-1. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Proteomics tools reveal startlingly high amounts of oxytocin in plasma and serum

    PubMed Central

    Brandtzaeg, Ole Kristian; Johnsen, Elin; Roberg-Larsen, Hanne; Seip, Knut Fredrik; MacLean, Evan L.; Gesquiere, Laurence R.; Leknes, Siri; Lundanes, Elsa; Wilson, Steven Ray

    2016-01-01

    The neuropeptide oxytocin (OT) is associated with a plethora of social behaviors, and is a key topic at the intersection of psychology and biology. However, tools for measuring OT are still not fully developed. We describe a robust nano liquid chromatography-mass spectrometry (nanoLC-MS) platform for measuring the total amount of OT in human plasma/serum. OT binds strongly to plasma proteins, but a reduction/alkylation (R/A) procedure breaks this bond, enabling ample detection of total OT. The method (R/A + robust nanoLC-MS) was used to determine total OT plasma/serum levels to startlingly high concentrations (high pg/mL-ng/mL). Similar results were obtained when combining R/A and ELISA. Compared to measuring free OT, measuring total OT can have advantages in e.g. biomarker studies. PMID:27528413

  13. Proteomics tools reveal startlingly high amounts of oxytocin in plasma and serum

    NASA Astrophysics Data System (ADS)

    Brandtzaeg, Ole Kristian; Johnsen, Elin; Roberg-Larsen, Hanne; Seip, Knut Fredrik; Maclean, Evan L.; Gesquiere, Laurence R.; Leknes, Siri; Lundanes, Elsa; Wilson, Steven Ray

    2016-08-01

    The neuropeptide oxytocin (OT) is associated with a plethora of social behaviors, and is a key topic at the intersection of psychology and biology. However, tools for measuring OT are still not fully developed. We describe a robust nano liquid chromatography-mass spectrometry (nanoLC-MS) platform for measuring the total amount of OT in human plasma/serum. OT binds strongly to plasma proteins, but a reduction/alkylation (R/A) procedure breaks this bond, enabling ample detection of total OT. The method (R/A + robust nanoLC-MS) was used to determine total OT plasma/serum levels to startlingly high concentrations (high pg/mL-ng/mL). Similar results were obtained when combining R/A and ELISA. Compared to measuring free OT, measuring total OT can have advantages in e.g. biomarker studies.

  14. Affinity proteomics reveals elevated muscle proteins in plasma of children with cerebral malaria.

    PubMed

    Bachmann, Julie; Burté, Florence; Pramana, Setia; Conte, Ianina; Brown, Biobele J; Orimadegun, Adebola E; Ajetunmobi, Wasiu A; Afolabi, Nathaniel K; Akinkunmi, Francis; Omokhodion, Samuel; Akinbami, Felix O; Shokunbi, Wuraola A; Kampf, Caroline; Pawitan, Yudi; Uhlén, Mathias; Sodeinde, Olugbemiro; Schwenk, Jochen M; Wahlgren, Mats; Fernandez-Reyes, Delmiro; Nilsson, Peter

    2014-04-01

    Systemic inflammation and sequestration of parasitized erythrocytes are central processes in the pathophysiology of severe Plasmodium falciparum childhood malaria. However, it is still not understood why some children are more at risks to develop malaria complications than others. To identify human proteins in plasma related to childhood malaria syndromes, multiplex antibody suspension bead arrays were employed. Out of the 1,015 proteins analyzed in plasma from more than 700 children, 41 differed between malaria infected children and community controls, whereas 13 discriminated uncomplicated malaria from severe malaria syndromes. Markers of oxidative stress were found related to severe malaria anemia while markers of endothelial activation, platelet adhesion and muscular damage were identified in relation to children with cerebral malaria. These findings suggest the presence of generalized vascular inflammation, vascular wall modulations, activation of endothelium and unbalanced glucose metabolism in severe malaria. The increased levels of specific muscle proteins in plasma implicate potential muscle damage and microvasculature lesions during the course of cerebral malaria.

  15. Proteomic Characterization of Zinc-Binding Proteins of Canine Seminal Plasma.

    PubMed

    Mogielnicka-Brzozowska, M; Kowalska, N; Fraser, L; Kordan, W

    2015-12-01

    The zinc-binding proteins (ZnBPs) of the seminal plasma are implicated in different processes related to sperm-egg fusion. The aim of this study was to characterize the ZnBPs of canine seminal plasma using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry. The ZnBPs were isolated from the ejaculates of five dogs by affinity chromatography and subjected to 2D-PAGE analysis. The acquired spots, detected across the gels, were analysed by mass spectrometry. Using 2D-PAGE analysis, it was shown that canine seminal plasma comprised about 46-57 zinc-binding polypeptides, with molecular mass ranging from 9.3 to 138.7 kDa and pI at pH 5.2-10.0. It was found that zinc-binding polypeptides of low molecular masses (9.3-19.0 kDa and pI at pH 6.1-10.0) were predominant in the seminal plasma, and seven polypeptides, with molecular masses ranging from 11.7 to 15.4 kDa and pI at pH 6.8-8.7, were characterized by high optical density values. In addition, analysis with mass spectrometry (LC-MS-MS/MS) revealed that the identified seven polypeptides are canine prostate-specific esterase (CPSE), which is the main proteolytic enzyme of the seminal plasma. The findings of this study indicate an important regulatory role of seminal plasma zinc ions in the functional activity of CPSE, which is of great significance for maintaining the normal function of canine prostate and the spermatozoa functions.

  16. MAPU: Max-Planck Unified database of organellar, cellular, tissue and body fluid proteomes

    PubMed Central

    Zhang, Yanling; Zhang, Yong; Adachi, Jun; Olsen, Jesper V.; Shi, Rong; de Souza, Gustavo; Pasini, Erica; Foster, Leonard J.; Macek, Boris; Zougman, Alexandre; Kumar, Chanchal; Wiśniewski, Jacek R.; Jun, Wang; Mann, Matthias

    2007-01-01

    Mass spectrometry (MS)-based proteomics has become a powerful technology to map the protein composition of organelles, cell types and tissues. In our department, a large-scale effort to map these proteomes is complemented by the Max-Planck Unified (MAPU) proteome database. MAPU contains several body fluid proteomes; including plasma, urine, and cerebrospinal fluid. Cell lines have been mapped to a depth of several thousand proteins and the red blood cell proteome has also been analyzed in depth. The liver proteome is represented with 3200 proteins. By employing high resolution MS and stringent validation criteria, false positive identification rates in MAPU are lower than 1:1000. Thus MAPU datasets can serve as reference proteomes in biomarker discovery. MAPU contains the peptides identifying each protein, measured masses, scores and intensities and is freely available at using a clickable interface of cell or body parts. Proteome data can be queried across proteomes by protein name, accession number, sequence similarity, peptide sequence and annotation information. More than 4500 mouse and 2500 human proteins have already been identified in at least one proteome. Basic annotation information and links to other public databases are provided in MAPU and we plan to add further analysis tools. PMID:17090601

  17. An improved candidate reference method for serum potassium measurement by inductively coupled plasma-mass spectrometry.

    PubMed

    Zhang, Rui; Ma, Huaian; Li, Huiling; Xu, Jing; Zhang, Jianping; Zhang, Yang; Wang, Qingtao

    2013-05-01

    In 2002, the National Institute of Standards and Technology (NIST) established a reference method for serum potassium based on inductively coupled plasma-mass spectrometry (ICP-MS). The aim of this study was to develop an inexpensive and improved candidate reference method for accurate and precise determination of serum potassium. Serum samples were diluted with 1% HNO3 supplemented with (59)Cobalt as isotope internal standard, and potassium was measured by ICP-MS. The new method was evaluated with NIST standard reference materials (SRMs), according to the Clinical and Laboratory Standard Institute's evaluation protocols. At 4.300 and 4.678 mmol/l levels, the present method demonstrated analytical imprecision of 0.09% and 0.14%, and recoveries of 99.67% to 99.88%, respectively. The bias between the target values of SRMs were -0.02% to +0.28%, respectively. This method was linear between 0.0000 and 6.87 mmol/l (R(2)=1.000). The method had an uncertainty (U95%) of 0.76%. The proposed ICP-MS method to measure serum potassium is precise and accurate, with high sensitivity and specificity. It may be considered as a candidate reference method for the determination of serum potassium. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. [Reference values for the blood coagulation tests in Mexico: usefulness of the pooled plasma from blood donors].

    PubMed

    Calzada-Contreras, Adriana; Moreno-Hernández, Manuel; Castillo-Torres, Noemi Patricia; Souto-Rosillo, Guadalupe; Hernández-Juárez, Jesús; Ricardo-Moreno, María Tania; Sánchez-Fernández, Maria Guadalupe de Jesús; García-González, América; Majluf-Cruz, Abraham

    2012-01-01

    The blood coagulation system maintains the blood in a liquid state and bleeding and thrombosis are the manifestations of its malfunction. Blood coagulation laboratory evaluates the physiology of this system. To establish both, the reference values for several tests performed at the blood coagulation laboratory as well as the utility of the pooled plasma to perform these assays. MATERIAL AND: In this descriptive, cross-sectional, randomized study, we collected plasma from Mexican Mestizos. Each pooled plasma was prepared with the plasma from at least 20 blood donors. We performed screening and special tests and the Levey-Jennings graphs were built and interpreted after each pass. Results of the tests were analyzed and their distribution was established using the Kolmogorov-Smirnov test. To establish the reference values we used 95% confidence intervals. We collected 72 pooled plasmas. The distribution for PT, APTT, and TT tests was abnormal. Although the PT test showed a bimodal distribution it was normal for factor VII. The reference values for the hemostatic, anticoagulant, and fibrinolytic factors were different from those suggested by the manufacturers. We established the reference values for the blood coagulation tests in the adult Mexican population. We have shown that the pooled plasma must be used for the screening tests. We suggest that each clinical laboratory should establish its own reference values (at least for the screening tests). To reach this objective, we encourage the use of the pooled plasma.

  19. Plasma proteomics shows an elevation of the anti-inflammatory protein APOA-IV in chronic equine laminitis

    PubMed Central

    2012-01-01

    Background Equine laminitis is a devastating disease that causes severe pain in afflicted horses and places a major economic burden on the horse industry. In acute laminitis, the disintegration of the dermal-epidermal junction can cause the third phalanx to detach from the hoof wall, leaving the horse unable to bear weight on the affected limbs. Horses that survive the acute phase transition into a chronic form of laminitis, which is often termed “founder”. Some evidence suggests that chronic laminar inflammation might be associated with alterations in the endocrine and immune systems. We investigated this broad hypothesis by using DIGE to assess global differences in the plasma proteome between horses with chronic laminitis and controls. Results We identified 16 differentially expressed proteins; the majority of these were involved in the interrelated coagulation, clotting, and kininogen cascades. Clinical testing of functional coagulation parameters in foundered horses revealed a slight delay in prothrombin (PT) clotting time, although most other indices were within normal ranges. Upregulation of the intestinal apolipoprotein APOA-IV in horses with chronic laminitis was confirmed by western blot. Conclusions Our results support the hypothesis that localized laminar inflammation may be linked to systemic alterations in immune regulation, particularly in the gastrointestinal system. Gastrointestinal inflammation has been implicated in the development of acute laminitis but has not previously been associated with chronic laminitis. PMID:23016951

  20. High-throughput proteomic characterization of plasma rich in growth factors (PRGF-Endoret)-derived fibrin clot interactome.

    PubMed

    Anitua, Eduardo; Prado, Roberto; Azkargorta, Mikel; Rodriguez-Suárez, Eva; Iloro, Ibon; Casado-Vela, Juan; Elortza, Felix; Orive, Gorka

    2015-11-01

    Plasma rich in growth factors (PRGF®-Endoret®) is an autologous technology that contains a set of proteins specifically addressed to wound healing and tissue regeneration. The scaffold formed by using this technology is a clot mainly composed of fibrin protein, forming a three-dimensional (3D) macroscopic network. This biomaterial is easily obtained by biotechnological means from blood and can be used in a range of situations to help wound healing and tissue regeneration. Although the main constituent of this clot is the fibrin scaffold, little is known about other proteins interacting in this clot that may act as adjuvants in the healing process. The aim of this study was to characterize the proteins enclosed by PRGF-Endoret scaffold, using a double-proteomic approach that combines 1D-SDS-PAGE approach followed by LC-MS/MS, and 2-DE followed by MALDI-TOF/TOF. The results presented here provide a description of the catalogue of key proteins in close contact with the fibrin scaffold. The obtained lists of proteins were grouped into families and networks according to gene ontology. Taken together, an enrichment of both proteins and protein families specifically involved in tissue regeneration and wound healing has been found. Copyright © 2013 John Wiley & Sons, Ltd.

  1. Comparison of plasma membrane proteomic changes of Arabidopsis suspension-cultured cells (T87 Line) after cold and ABA treatment in association with freezing tolerance development.

    PubMed

    Li, Bin; Takahashi, Daisuke; Kawamura, Yukio; Uemura, Matsuo

    2012-03-01

    The plasma membrane (PM) is the primary site of freezing injury in plants. To determine global changes in PM protein profiles in association with freezing tolerance development, proteome analysis of the purified PM of Arabidopsis suspension-cultured cells (T87 line) was conducted with label-free protein quantification technology. Freezing tolerance of Arabidopsis cells at the lag growth phase (8 d old) increased after cold acclimation (CA) or ABA treatment. Proteome analysis assigned 658 proteins in the PM in total, of which 45.3% (298 proteins) were predicted to have transmembrane domains. They were classified into several functional categories, with the primary categories being proteins in transporters, signal transduction, protein destination and storage, and cell structure. After CA, 271 proteins increased and 111 proteins decreased. ABA treatment resulted in 185 increased and 56 decreased proteins. Of these, 139 increased and 49 decreased proteins were identified in common after both CA and ABA treatment. In addition, there were proteins specifically expressed in cold- (132 increased and 62 decreased) or ABA- (46 increased and 7 decreased) treated cells. Collectively, our results clearly show that (i) responses of the PM proteome to CA and ABA treatment overlap substantially but, at the same time, some proteins exhibited different response patterns in each treatment; and (ii) the majority of ABA-responsive proteins are CA-responsive proteins but not vice versa, suggesting complex interactions of CA and ABA signaling pathways in the PM proteome responses.

  2. Hematological and plasma biochemical reference ranges of Alaskan seabirds: Their ecological significance and clinical importance

    USGS Publications Warehouse

    Newman, S.H.; Piatt, J.F.; White, J.

    1997-01-01

    Blood was analyzed from 151 pelagic marine birds to establish reference ranges for hematological and plasma biochemical parameters from healthy, wild populations of Pacific seabirds. Of the 13 species examined, 9 were from the Family Alcidae (N = 122 individuals) and the remainder (N = 29) from the Families Phalacrocoracidae, Laridae, and Procellariidae. Three of 8 hematological parameters (total white blood cell count, lymphocyte count and eosinophil count) differed significantly among species, as did 9 of 13 plasma biochemical parameters (alkaline phosphatase, aspartate aminotransferase, creatine kinase, cholesterol, glucose, lactate dehydrogenase, total bilirubin, total protein and field total protein). There were no differences among species for packed cell volume, buffy coat, cell counts of heterophils, monocytes and basophils, or for concentrations of alanine aminotransferase, triglycerides, uric acid and calcium. Plasma calcium concentration, triglyceride levels and field total protein varied significantly between sexes, with females having higher mean concentrations of all 3 parameters. However, no significant relationships between measures of breeding condition (brood patch size, subcutaneous and mesenteric fat deposits, or ovarian follicle size and ovary weight) and calcium or alkaline phosphatase concentrations in female birds could be identified. Alanine aminotransferase and uric acid were the only analytes which did not differ significantly between species or sexes.

  3. Affinity Proteomics Reveals Elevated Muscle Proteins in Plasma of Children with Cerebral Malaria

    PubMed Central

    Pramana, Setia; Conte, Ianina; Brown, Biobele J.; Orimadegun, Adebola E.; Ajetunmobi, Wasiu A.; Afolabi, Nathaniel K.; Akinkunmi, Francis; Omokhodion, Samuel; Akinbami, Felix O.; Shokunbi, Wuraola A.; Kampf, Caroline; Pawitan, Yudi; Uhlén, Mathias; Sodeinde, Olugbemiro; Schwenk, Jochen M.; Wahlgren, Mats; Fernandez-Reyes, Delmiro; Nilsson, Peter

    2014-01-01

    Systemic inflammation and sequestration of parasitized erythrocytes are central processes in the pathophysiology of severe Plasmodium falciparum childhood malaria. However, it is still not understood why some children are more at risks to develop malaria complications than others. To identify human proteins in plasma related to childhood malaria syndromes, multiplex antibody suspension bead arrays were employed. Out of the 1,015 proteins analyzed in plasma from more than 700 children, 41 differed between malaria infected children and community controls, whereas 13 discriminated uncomplicated malaria from severe malaria syndromes. Markers of oxidative stress were found related to severe malaria anemia while markers of endothelial activation, platelet adhesion and muscular damage were identified in relation to children with cerebral malaria. These findings suggest the presence of generalized vascular inflammation, vascular wall modulations, activation of endothelium and unbalanced glucose metabolism in severe malaria. The increased levels of specific muscle proteins in plasma implicate potential muscle damage and microvasculature lesions during the course of cerebral malaria. PMID:24743550

  4. Differential proteomic profiling of endometrium and plasma indicate the importance of hydrolysis in bovine endometritis.

    PubMed

    Zhang, Shi-Dong; Dong, Shu-Wei; Wang, Dong-Sheng; Oguejiofor, Chike F; Fouladi-Nashta, Ali A; Yang, Zhi-Qiang; Yan, Zuo-Ting

    2017-09-13

    Endometritis is an important disease of dairy cows that leads to significant economic losses in the dairy cattle industry. To investigate the alteration of proteins associated with endometritis in the dairy cow, the isobaric tags for relative and absolute quantification (iTRAQ) technique was applied to quantitatively identify differentially expressed proteins (DEP) in the endometrium and peripheral plasma of Chinese Holstein cows with endometritis. Compared with the normal (control) group, 159 DEP in the endometrium and 137 DEP in the plasma were identified in cows with endometritis. Gene ontology analysis demonstrated that the predominant endometrial DEP were primarily involved in responses to stimulus and stress processes and mainly played a role in hydrolysis in the extracellular region. The predominant plasma DEP were mainly components of the cytosol and non-membrane-bound organelles, and they were involved in the response to stress and regulation of enzyme activity. Protein-protein interaction of tissue DEP revealed that some core seed proteins, such as RAC2, ITGB2, and CDH1 in the same network as CD14, MMP3, and MMP9, had important functions in the cross-talk of pathways related to extracellular proteolysis. In summary, significant enzymatic hydrolase activity in the extracellular region is proposed as a molecular mechanism by which altered proteins may promote inflammation and hence endometritis. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  7. Enrichment and preparation of plasma membrane proteins from Arabidopsis thaliana for global proteomic analysis using liquid chromatography-tandem mass spectrometry.

    PubMed

    Mitra, Srijeet K; Clouse, Steven D; Goshe, Michael B

    2009-01-01

    The plasma membrane proteins are critical components in cellular control and differentiation and thus are of special interest to those studying signal transduction mechanisms in all organisms. When conducting proteomic studies on membrane components of cells and tissues, the complexity is not simply confined to the large number of proteins present in the sample but also to the highly hydrophobic nature of membrane proteins containing multiple transmembrane domains. Consequently, these proteins are more difficult to analyze by mass spectrometry, particularly if protein sequence coverage is to be established. This chapter contains a method for extraction, solubilization, alkylation, proteolysis, and identification of hydrophobic integral plasma membrane proteins for large-scale proteomic analysis using strong cation exchange chromatography (SCXC) and liquid chromatography-tandem mass spectrometry (LC/MS/MS). In our approach, microsomes are isolated from plant tissue and then subjected to a two-phase extraction procedure to enrich for plasma membranes. Proteins are extracted and solubilized from the membrane using a methanol-aqueous buffer system that allows for effective reduction, cysteinyl alkylation, and tryptic digestion for subsequent SCXC-LC/MS/MS analysis. Our protocol is also amenable to isotope labeling methods to quantify integral membrane protein expression and post-translational modifications. In addition to plants, the method can be applied to other systems quite readily; thus, we anticipate that it will be of general interest to those characterizing plasma membrane proteins of any organism.

  8. Plasma proteomics of lung cancer by a linkage of multi-dimensional liquid chromatography and two-dimensional difference gel electrophoresis.

    PubMed

    Okano, Tetsuya; Kondo, Tadashi; Kakisaka, Tatsuhiko; Fujii, Kiyonaga; Yamada, Masayo; Kato, Harubumi; Nishimura, Toshihide; Gemma, Akihiko; Kudoh, Shoji; Hirohashi, Setsuo

    2006-07-01

    To investigate aberrant plasma proteins in lung cancer, we compared the proteomic profiles of serum from five lung cancer patients and from four healthy volunteers. Immuno-affinity chromatography was used to deplete highly abundant plasma proteins, and the resulting plasma samples were separated into eight fractions by anion-exchange chromatography. Quantitative protein profiles of the fractionated samples were generated by two-dimensional difference gel electrophoresis, in which the experimental samples and the internal control samples were labeled with different dyes and co-separated by two-dimensional polyacrylamide gel electrophoresis. This approach succeeded in resolving 3890 protein spots. For 364 of the protein spots, the expression level in lung cancer was more than twofold different from that in the healthy volunteers. These differences were statistically significant (Student's t-test, p-value less than 0.05). Mass spectrometric protein identification revealed that the 364 protein spots corresponded to 58 gene products, including the classical plasma proteins and the tissue-leakage proteins catalase, clusterin, ficolin, gelsolin, lumican, tetranectin, triosephosphate isomerase and vitronectin. The combination of multi-dimensional liquid chromatography and two-dimensional difference gel electrophoresis provides a valuable tool for serum proteomics in lung cancer.

  9. Determination of inflammatory and prominent proteomic changes in plasma and adipose tissue after high-intensity intermittent training in overweight and obese males

    PubMed Central

    Leggate, Melanie; Carter, Wayne G.; Evans, Matthew J. C.; Vennard, Rebecca A.; Sribala-Sundaram, Sarah

    2012-01-01

    This study aimed to determine whether 2 wk of high-intensity intermittent training (HIIT) altered inflammatory status in plasma and adipose tissue in overweight and obese males. Twelve participants [mean (SD): age 23.7 (5.2) yr, body mass 91.0 (8.0) kg, body mass index 29.1 (3.1) kg/m2] undertook six HIIT sessions over 2 wk. Resting blood and subcutaneous abdominal adipose tissue samples were collected and insulin sensitivity determined, pre- and posttraining. Inflammatory proteins were quantified in plasma and adipose tissue. There was a significant decrease in soluble interleukin-6 receptor (sIL-6R; P = 0.050), monocyte chemotactic protein-1 (MCP-1, P = 0.047), and adiponectin (P = 0.041) in plasma posttraining. Plasma IL-6, intercellular adhesion molecule-1 (ICAM-1), tumor necrosis factor-α (TNF-α), IL-10, and insulin sensitivity did not change. In adipose tissue, IL-6 significantly decreased (P = 0.036) and IL-6R increased (P = 0.037), while adiponectin tended to decrease (P = 0.056), with no change in ICAM-1 posttraining. TNF-α, MCP-1, and IL-10 were not detectable in adipose tissue. Adipose tissue homogenates were then resolved using one-dimensional gel electrophoresis, and major changes in the adipose tissue proteome, as a consequence of HIIT, were evaluated. This proteomic approach identified significant reductions in annexin A2 (P = 0.046) and fatty acid synthase (P = 0.016) as a response to HIIT. The present investigation suggests 2 wk of HIIT is sufficient to induce beneficial alterations in the resting inflammatory profile and adipose tissue proteome of an overweight and obese male cohort. PMID:22267387

  10. Markers of vascular disease in plasma from patients with chronic kidney disease identified by proteomic analysis.

    PubMed

    Schiffer, Eric; Liabeuf, Sophie; Lacroix, Chrystelle; Temmar, Mohamed; Renard, Cedric; Monsarrat, Bernard; Choukroun, Gabriel; Lemke, Horst-Dieter; Vanholder, Raymond; Mischak, Harald; Massy, Ziad A

    2011-04-01

    Chronic kidney disease (CKD) patients belong to the group of patients with a high prevalence of cardiovascular disease (CVD). Arterial calcification and aortic stiffness are currently used as surrogates for vascular alterations. However, still little is known about prediction and the patho-physiologic mechanisms leading to CVD. We applied capillary electrophoresis coupled mass spectrometry profiling to blood specimens collected from 34 CKD stage 5D patients suffering from vascular alterations to allow insights into the molecular pathology of the disease. Statistical comparison of plasma profiles from mild and severe CVD cases according to either arterial calcification or aortic stiffness unveiled 13 novel biomarkers for vascular disease. Tandem mass spectrometry identified four of these as fragments of collagen alpha-1 type I and III and one as fragment of apolipoprotein CIII. Integrated in a distinct pattern the candidates were validated using the moderate CVD cases among the 34 CKD patients (N=11) and an additional independent blinded cohort of CKD stage 4-5 patients (N=21), who all had not been considered during biomarker discovery. The panel distinguished mild and severe CVD with sensitivity of 89% and specificity of 67% in this independent cohort. This diagnostic phase I/II study supports the notion that vascular alterations are reflected by distinct changes in plasma profiles of CKD patients.

  11. Plasma motor/generator reference system designs for power and propulsion

    NASA Technical Reports Server (NTRS)

    Mccoy, James E.

    1987-01-01

    Four Plasma Motor/Generator (PMG) Reference Systems, hollow cathode-based versions of the electrodynamic tether concept which are to be used in study and analysis of future propulsion and power applications, are discussed. These systems are equally applicable for use as electric generators to provide power to a spacecraft or as electric motors using power from the spacecraft. Operating at relatively high current and low voltage, the PMGs avoid requirements for technological advances to handle very high voltages. Permanent deployment with passive I x B control of tether dynamics eliminates the complexity and weight of a TSS style tether reel. A 20 kW PMG uses 10 km of number two aluminum wire, weighs 1200 kg, and has an electrical efficiency of 93 percent. A larger 200 kW system uses 20 km of number 00 aluminum wire, weighs 4200 kg, and operates at 87 percent efficiency.

  12. Effect of embryonic development on the chicken egg yolk plasma proteome after 12 days of incubation.

    PubMed

    Réhault-Godbert, Sophie; Mann, Karlheinz; Bourin, Marie; Brionne, Aurélien; Nys, Yves

    2014-03-26

    To better appreciate the dynamics of yolk proteins during embryonic development, we analyzed the protein quantitative changes occurring in the yolk plasma at the day of lay and after 12 days of incubation, by comparing unfertilized and fertilized chicken eggs. Of the 127 identified proteins, 69 showed relative abundance differences among conditions. Alpha-fetoprotein and two uncharacterized proteins (F1NHB8 and F1NMM2) were identified for the first time in the egg. After 12 days of incubation, five proteins (vitronectin, α-fetoprotein, similar to thrombin, apolipoprotein B, and apovitellenin-1) showed a major increase in relative abundance, whereas 15 proteins showed a significant decrease in the yolks of fertilized eggs. In unfertilized/table eggs, we observed an accumulation of proteins likely to originate from other egg compartments during incubation. This study provides basic knowledge on the utilization of egg yolk proteins by the embryo and gives some insight into how storage can affect egg quality.

  13. Proteomic analysis of the plasma membrane-movement tubule complex of cowpea mosaic virus.

    PubMed

    den Hollander, Paulus W; de Sousa Geraldino Duarte, Priscilla; Bloksma, Hanke; Boeren, Sjef; van Lent, Jan W M

    2016-05-01

    Cowpea mosaic virus forms tubules constructed from the movement protein (MP) in plasmodesmata (PD) to achieve cell-to-cell movement of its virions. Similar tubules, delineated by the plasma membrane (PM), are formed protruding from the surface of infected protoplasts. These PM-tubule complexes were isolated from protoplasts by immunoprecipitation and analysed for their protein content by tandem mass spectrometry to identify host proteins with affinity for the movement tubule. Seven host proteins were abundantly present in the PM-tubule complex, including molecular chaperonins and an AAA protein. Members of both protein families have been implicated in establishment of systemic infection. The potential role of these proteins in tubule-guided cell-cell transport is discussed.

  14. Hematologic and plasma biochemistry reference intervals of healthy adult barn owls (Tyto alba).

    PubMed

    Szabo, Zoltan; Klein, Akos; Jakab, Csaba

    2014-06-01

    Hematologic and plasma biochemistry parameters of barn owls (Tyto alba) were studied in collaboration by the Exotic Division of the Faculty of Veterinary Science of the Szent Istvan University and the Eötvös Loránd University, both in Budapest, Hungary. Blood samples were taken from a total of 42 adult barn owls kept in zoos and bird repatriation stations. The following quantitative and qualitative hematologic values were determined: packed cell volume, 46.2 +/- 4%; hemoglobin concentration, 107 +/- 15 g/L; red blood cell count, 3.2 +/- 0.4 x 10(12)/L; white blood cell count, 13.7 +/- 2.7 x 10(9)/L; heterophils, 56.5 +/- 11.5% (7.8 +/- 2 x 10(9)/L); lymphocytes, 40.3 +/- 10.9% (5.5 +/- 1.9 x 10(9)/L); monocytes, 1.8 +/- 2.1% (0.3 +/- 0.3 x 10(9)/ L); eosinophils, 1 +/- 1% (0.1 +/- 0.1 x 10(9)/L); and basophils, 0.6 +/- 0.5% (0.1 +/- 0.1 x 10(9)/L). The following plasma biochemistry values also were determined: aspartate aminotransferase, 272 +/- 43 U/L; L-gamma-glutamyltransferase, 9.5 +/- 4.7 U/L; lipase, 31.7 +/- 11.1 U/L; creatine kinase, 2228 +/- 578 U/L; lactate dehydrogenase, 1702 +/- 475 U/L; alkaline phosphatase, 358 +/- 197 U/L; amylase, 563 +/- 114 U/L; glutamate dehydrogenase, 7.5 +/- 2.5 U/L; total protein, 30.6 +/- 5.3 g/L; uric acid, 428 +/- 102 micromol/L; and bile acids, 43 +/- 18 micromol/L. These results provide reliable reference values for the clinical interpretation of hematologic and plasma biochemistry results for the species.

  15. Proteomic Characterization of Pig Sperm Anterior Head Plasma Membrane Reveals Roles of Acrosomal Proteins in ZP3 Binding.

    PubMed

    Kongmanas, Kessiri; Kruevaisayawan, Hathairat; Saewu, Arpornrad; Sugeng, Clarissa; Fernandes, Jason; Souda, Puneet; Angel, Jonathan B; Faull, Kym F; Aitken, R John; Whitelegge, Julian; Hardy, Daniel; Berger, Trish; Baker, Mark A; Tanphaichitr, Nongnuj

    2015-02-01

    The sperm anterior head plasma membrane (APM) is the site where sperm first bind to the zona pellucida (ZP). This binding reaches the maximum following the sperm capacitation process. To gain a better understanding of the sperm-ZP binding mechanisms, we compared protein profiles obtained from mass spectrometry of APM vesicles isolated from non-capacitated and capacitated sperm. The results revealed that ZP-binding proteins were the most abundant group of proteins, with a number of them showing increased levels in capacitated sperm. Blue native gel electrophoresis and far-western blotting revealed presence of high molecular weight (HMW) protein complexes in APM vesicles of both non-capacitated and capacitated sperm, but the complexes (∼750-1300 kDa) from capacitated sperm possessed much higher binding capacity to pig ZP3 glycoprotein. Proteomic analyses indicated that a number of proteins known for their acrosome localization, including zonadhesin, proacrosin/acrosin and ACRBP, were components of capacitated APM HMW complexes, with zonadhesin being the most enriched protein. Our immunofluorescence results further demonstrated that a fraction of these acrosomal proteins was transported to the surface of live acrosome-intact sperm during capacitation. Co-immunoprecipitation indicated that zonadhesin, proacrosin/acrosin and ACRBP interacted with each other and they may traffic as a complex from the acrosome to the sperm surface. Finally, the significance of zonadhesin in the binding of APM HMW complexes to pig ZP3 was demonstrated; the binding ability was decreased following treatment of the complexes with anti-zonadhesin antibody. Our results suggested that acrosomal proteins, especially zonadhesin, played roles in the initial sperm-ZP binding during capacitation.

  16. Plasma metabolomics and proteomics profiling after a postprandial challenge reveal subtle diet effects on human metabolic status.

    PubMed

    Pellis, Linette; van Erk, Marjan J; van Ommen, Ben; Bakker, Gertruud C M; Hendriks, Henk F J; Cnubben, Nicole H P; Kleemann, Robert; van Someren, Eugene P; Bobeldijk, Ivana; Rubingh, Carina M; Wopereis, Suzan

    2012-04-01

    We introduce the metabolomics and proteomics based Postprandial Challenge Test (PCT) to quantify the postprandial response of multiple metabolic processes in humans in a standardized manner. The PCT comprised consumption of a standardized 500 ml dairy shake containing respectively 59, 30 and 12 energy percent lipids, carbohydrates and protein. During a 6 h time course after PCT 145 plasma metabolites, 79 proteins and 7 clinical chemistry parameters were quantified. Multiple processes related to metabolism, oxidation and inflammation reacted to the PCT, as demonstrated by changes of 106 metabolites, 31 proteins and 5 clinical chemistry parameters. The PCT was applied in a dietary intervention study to evaluate if the PCT would reveal additional metabolic changes compared to non-perturbed conditions. The study consisted of a 5-week intervention with a supplement mix of anti-inflammatory compounds in a crossover design with 36 overweight subjects. Of the 231 quantified parameters, 31 had different responses over time between treated and control groups, revealing differences in amino acid metabolism, oxidative stress, inflammation and endocrine metabolism. The results showed that the acute, short term metabolic responses to the PCT were different in subjects on the supplement mix compared to the controls. The PCT provided additional metabolic changes related to the dietary intervention not observed in non-perturbed conditions. Thus, a metabolomics based quantification of a standardized perturbation of metabolic homeostasis is more informative on metabolic status and subtle health effects induced by (dietary) interventions than quantification of the homeostatic situation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0320-5) contains supplementary material, which is available to authorized users.

  17. A Combinatorial Peptide Ligand Libraries Treatment Followed by a Dual-Enzyme, Dual-Activation Approach on a nano-flow LC/Orbitrap/ETD for Comprehensive Analysis of Swine Plasma Proteome

    PubMed Central

    Tu, Chengjian; Li, Jun; Young, Rebeccah; Page, Brian J.; Engler, Frank; Halfon, Marc S.; Canty, John M.; Qu, Jun

    2011-01-01

    The plasma proteome holds enormous clinical potentials, yet an in-depth analysis of the plasma proteome remains a daunting challenge due to its high complexity and the extremely-wide dynamic range in protein concentrations. Furthermore, existing antibody-based approaches for depleting high-abundance proteins are not adaptable to the analysis of animal plasma proteome, which are often essential for experimental pathology/pharmacology. Here we describe a highly-comprehensive method for the investigation of animal plasma proteomes, which employs an optimized combinatorial peptide ligand libraries (CPLL) treatment to reduce the protein concentration dynamic range and a dual-enzyme, dual-activation strategy to achieve high proteomic coverage. The CPLL-treatment enriched the lower-abundance proteins by >100-fold when loading the samples in moderately-denaturing condition with multiple loading-washing cycles. The native and the CPLL-treated plasma were digested in-parallel respectively by two enzymes (trypsin and GluC) carrying orthogonal specificities. By performing this differential proteolysis, the proteome coverage is improved where peptides produced by only one enzyme are poorly detectable. Digests were fractionated with high-resolution SCX chromatography and then resolved on a long, heated nano-LC column. MS analysis was performed on an LTQ/Orbitrap respectively with two complementary activation methods (CID and ETD). We applied this optimized strategy to investigate the plasma proteome from swine, a prominent animal model for cardiovascular diseases(CVD). This large-scale analysis results in an identification of a total 3421 unique proteins, spanning a concentration range of 9–10 orders of magnitude. The proteins were identified under a set of commonly-accepted criteria including precursor mass error<15 ppm, Xcorr cutoffs, ≥two unique peptides at the peptide probability≥95% and protein probability≥99%, and the peptide FDR of the dataset was 1.8% as

  18. Gas and heat dynamics of a micro-scaled atmospheric pressure plasma reference jet

    NASA Astrophysics Data System (ADS)

    Kelly, Seán; Golda, Judith; Turner, Miles M.; Schulz-von der Gathen, Volker

    2015-11-01

    Gas and heat dynamics of the ‘Cooperation on Science and Technology (COST) Reference Microplasma Jet’ (COST-jet), a European lead reference device for low temperature atmospheric pressure plasma application, are investigated. Of particular interest to many biomedical application scenarios, the temperature characteristics of a surface impacted by the jet are revealed. Schlieren imaging, thermocouple measurements, infrared thermal imaging and numerical modelling are employed. Temperature spatial profiles in the gas domain reveal heating primarily of the helium fraction of the gas mixture. Thermocouple and model temporal data show a bounded exponential temperature growth described by a single characteristic time parameter to reach  ∼63% or (1-1/e) fraction of the temperature increase. Peak temperatures occurred in the gas domain where the carrier jet exits the COST-jet, with values ranging from ambient temperatures to in excess of 100 °C in ‘α-mode’ operation. In a horizontal orientation of the COST-jet a curved trajectory of the helium effluent at low gas flows results from buoyant forces. Gas mixture profiles reveal significant containment of the helium concentrations for a surface placed in close proximity to the COST-jet. Surface heating of a quartz plate follows a similar bounded exponential temporal temperature growth as device heating. Spatial profiles of surface heating are found to correlate strongly to the impacting effluent where peak temperatures occur in regions of maximum surface helium concentration.

  19. Differential diagnosis of azoospermia with proteomic biomarkers ECM1 and TEX101 quantified in seminal plasma.

    PubMed

    Drabovich, Andrei P; Dimitromanolakis, Apostolos; Saraon, Punit; Soosaipillai, Antoninus; Batruch, Ihor; Mullen, Brendan; Jarvi, Keith; Diamandis, Eleftherios P

    2013-11-20

    Male fertility problems range from diminished production of sperm, or oligozoospermia, to nonmeasurable levels of sperm in semen, or azoospermia, which is diagnosed in nearly 2% of men in the general population. Testicular biopsy is the only definitive diagnostic method to distinguish between obstructive (OA) and nonobstructive (NOA) azoospermia and to identify the NOA subtypes of hypospermatogenesis, maturation arrest and Sertoli cell-only syndrome. We measured by selected reaction monitoring assay 18 biomarker candidates in 119 seminal plasma samples from men with normal spermatogenesis and azoospermia, and identified two proteins, epididymis-expressed ECM1 and testis-expressed TEX101, which differentiated OA and NOA with high specificities and sensitivities. The performance of ECM1 was confirmed by enzyme-linked immunosorbent assay. On the basis of a cutoff level of 2.3 μg/ml derived from the current data, we could distinguish OA from normal spermatogenesis with 100% specificity, and OA from NOA with 73% specificity, at 100% sensitivity. Immunohistochemistry and an immunoenrichment mass spectrometry-based assay revealed the differential expression of TEX101 in distinct NOA subtypes. TEX101 semen concentrations differentiated Sertoli cell-only syndrome from the other categories of NOA. As a result, we propose a simple two-biomarker decision tree for the differential diagnosis of OA and NOA and, in addition, for the differentiation of NOA subtypes. Clinical assays for ECM1 and TEX101 have the potential to replace most of the diagnostic testicular biopsies and facilitate the prediction of outcome of sperm retrieval procedures, thus increasing the reliability and success of assisted reproduction techniques.

  20. A lectin-coupled, targeted proteomic mass spectrometry (MRM MS) platform for identification of multiple liver cancer biomarkers in human plasma.

    PubMed

    Ahn, Yeong Hee; Shin, Park Min; Oh, Na Ree; Park, Gun Wook; Kim, Hoguen; Yoo, Jong Shin

    2012-09-18

    Aberrantly glycosylated proteins related to liver cancer progression were captured with specific lectin and identified from human plasma by multiple reaction monitoring (MRM) mass spectrometry as multiple biomarkers for hepatocellular carcinoma (HCC). The lectin fractionation for fucosylated protein glycoforms in human plasma was conducted with a fucose-specific aleuria aurantia lectin (AAL). Following tryptic digestion of the lectin-captured fraction, plasma samples from 30 control cases (including 10 healthy, 10 hepatitis B virus [HBV], and 10 cirrhosis cases) and 10 HCC cases were quantitatively analyzed by MRM to identify which glycoproteins are viable HCC biomarkers. A1AG1, AACT, A1AT, and CERU were found to be potent biomarkers to differentiate HCC plasma from control plasmas. The AUROC generated independently from these four biomarker candidates ranged from 0.73 to 0.92. However, the lectin-coupled MRM assay with multiple combinations of biomarker candidates is superior statistically to those generated from the individual candidates with AUROC more than 0.95, which can be an alternative to the immunoassay inevitably requiring tedious development of multiple antibodies against biomarker candidates to be verified. Eventually the lectin-coupled, targeted proteomic mass spectrometry (MRM MS) platform was found to be efficient to identify multiple biomarkers from human plasma according to cancer progression. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. Plasma Asymmetric Dimethylarginine and Adverse Events in Patients with Atrial Fibrillation Referred for Coronary Angiogram

    PubMed Central

    Chao, Tze-Fan; Lu, Tse-Min; Lin, Yenn-Jiang; Tsao, Hsuan-Ming; Chang, Shih-Lin; Lo, Li-Wei; Hu, Yu-Feng; Tuan, Ta-Chuan; Hsieh, Ming-Hsiung; Chen, Shih-Ann

    2013-01-01

    Objectives Elevated plasma levels of asymmetric dimethylarginine (ADMA) have been reported to be associated with endothelial dysfunction, inflammation, and oxidative stress in multiple cardiovascular diseases. This study aimed to investigate whether ADMA was a predictor of clinical outcomes in atrial fibrillation (AF). Methods and Results From 2006-2009, 990 individuals were referred to our institution for coronary angiography. Among these patients, 141 subjects with a diagnosis of AF, including 52 paroxysmal AF (PAF) and 89 non-paroxysmal AF (non-PAF) patients, were identified as the study population. Plasma ADMA levels were measured. An adverse event was defined as the occurrence of ischemic stroke or cardiovascular death. The ADMA levels were higher in AF than non-AF patients (0.50±0.13 versus 0.45±0.07 µmol/L; p<0.001). Besides, non-PAF patients had higher ADMA levels than PAF patients (0.52±0.15 versus 0.48±0.08 µmol/L; p<0.001). During the follow-up of 30.7±14.4 months, 21 patients (14.9%) experienced adverse events, including cardiovascular death in 7 patients and ischemic stroke in 14. ADMA level, CHA2DS2-VASc score, and left atrial diameter were independent predictors of adverse events in the multivariate analysis. At a cutoff-value of 0.55 µmol/L, the Kaplan-Meier survival analysis showed that patients with a high ADMA level had a higher event rate during the follow-up period. Conclusions A higher level of ADMA was a risk factor of adverse events in AF patients, which was independent from the CHA2DS2-VASc score. It deserves to further study whether ADMA could potentially refine the clinical risk stratification in AF. PMID:23951217

  2. Reference intervals for plasma free metanephrines with an age adjustment for normetanephrine for optimized laboratory testing of phaeochromocytoma.

    PubMed

    Eisenhofer, Graeme; Lattke, Peter; Herberg, Maria; Siegert, Gabriele; Qin, Nan; Därr, Roland; Hoyer, Jana; Villringer, Arno; Prejbisz, Aleksander; Januszewicz, Andrzej; Remaley, Alan; Martucci, Victoria; Pacak, Karel; Ross, H Alec; Sweep, Fred C G J; Lenders, Jacques W M

    2013-01-01

    Measurements of plasma normetanephrine and metanephrine provide a useful diagnostic test for phaeochromocytoma, but this depends on appropriate reference intervals. Upper cut-offs set too high compromise diagnostic sensitivity, whereas set too low, false-positives are a problem. This study aimed to establish optimal reference intervals for plasma normetanephrine and metanephrine. Blood samples were collected in the supine position from 1226 subjects, aged 5-84 y, including 116 children, 575 normotensive and hypertensive adults and 535 patients in whom phaeochromocytoma was ruled out. Reference intervals were examined according to age and gender. Various models were examined to optimize upper cut-offs according to estimates of diagnostic sensitivity and specificity in a separate validation group of 3888 patients tested for phaeochromocytoma, including 558 with confirmed disease. Plasma metanephrine, but not normetanephrine, was higher (P < 0.001) in men than in women, but reference intervals did not differ. Age showed a positive relationship (P < 0.0001) with plasma normetanephrine and a weaker relationship (P = 0.021) with metanephrine. Upper cut-offs of reference intervals for normetanephrine increased from 0.47 nmol/L in children to 1.05 nmol/L in subjects over 60 y. A curvilinear model for age-adjusted compared with fixed upper cut-offs for normetanephrine, together with a higher cut-off for metanephrine (0.45 versus 0.32 nmol/L), resulted in a substantial gain in diagnostic specificity from 88.3% to 96.0% with minimal loss in diagnostic sensitivity from 93.9% to 93.6%. These data establish age-adjusted cut-offs of reference intervals for plasma normetanephrine and optimized cut-offs for metanephrine useful for minimizing false-positive results.

  3. Reference intervals for plasma free metanephrines with an age adjustment for normetanephrine for optimized laboratory testing of phaeochromocytoma

    PubMed Central

    Eisenhofer, Graeme; Lattke, Peter; Herberg, Maria; Siegert, Gabriele; Qin, Nan; Därr, Roland; Hoyer, Jana; Villringer, Arno; Prejbisz, Aleksander; Januszewicz, Andrzej; Remaley, Alan; Martucci, Victoria; Pacak, Karel; Ross, H Alec; Sweep, Fred C G J; Lenders, Jacques W M

    2016-01-01

    Background Measurements of plasma normetanephrine and metanephrine provide a useful diagnostic test for phaeochromocytoma, but this depends on appropriate reference intervals. Upper cut-offs set too high compromise diagnostic sensitivity, whereas set too low, false-positives are a problem. This study aimed to establish optimal reference intervals for plasma normetanephrine and metanephrine. Methods Blood samples were collected in the supine position from 1226 subjects, aged 5–84 y, including 116 children, 575 normotensive and hypertensive adults and 535 patients in whom phaeochromocytoma was ruled out. Reference intervals were examined according to age and gender. Various models were examined to optimize upper cut-offs according to estimates of diagnostic sensitivity and specificity in a separate validation group of 3888 patients tested for phaeochromocytoma, including 558 with confirmed disease. Results Plasma metanephrine, but not normetanephrine, was higher (P < 0.001) in men than in women, but reference intervals did not differ. Age showed a positive relationship (P < 0.0001) with plasma normetanephrine and a weaker relationship (P = 0.021) with metanephrine. Upper cut-offs of reference intervals for normetanephrine increased from 0.47 nmol/L in children to 1.05 nmol/L in subjects over 60 y. A curvilinear model for age-adjusted compared with fixed upper cut-offs for normetanephrine, together with a higher cut-off for metanephrine (0.45 versus 0.32 nmol/L), resulted in a substantial gain in diagnostic specificity from 88.3% to 96.0% with minimal loss in diagnostic sensitivity from 93.9% to 93.6%. Conclusions These data establish age-adjusted cut-offs of reference intervals for plasma normetanephrine and optimized cut-offs for metanephrine useful for minimizing false-positive results. PMID:23065528

  4. Hematologic, Plasma Biochemical, and Lipid Panel Reference Intervals in Orange-winged Amazon Parrots ( Amazona amazonica ).

    PubMed

    Vergneau-Grosset, Claire; Polley, Tamsen; Holt, Danielle Carrade; Vernau, William; Paul-Murphy, Joanne

    2016-12-01

    To establish reference intervals in orange-winged Amazon parrots ( Amazona amazonica ) for the complete blood count, plasma biochemical values, and lipid panel and to evaluate age- and sex-related variations, blood samples were obtained from 29 healthy juvenile and adult parrots. Concentrations of total protein, bile acids, phosphorus, total cholesterol, low-density-lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol were significantly higher in adult compared with juvenile birds, while uric acid concentration was significantly higher in juveniles. The white blood cell count, lymphocyte count, and phosphorus and potassium concentrations were significantly higher in females, while chloride concentration was significantly higher in males. In this species, direct measurement of LDL-C resulted in lower concentrations than LDL-C calculated with the Friedewald formula. Assessment of the agreement between the calculated and measured LDL-C concentrations indicated a systematic bias of 19.1 mg/dL and a proportional bias of 1.07. A correction factor of -19 mg/L could be applied to the Friedewald formula, to obtain a result closer to the measured LDL-C, providing clinically acceptable (<20% difference) agreement in 66% of the samples. Triglyceride concentrations within the range measured in healthy birds of the present study did not significantly affect the bias between calculated and directly measured LDL-C. Further studies are needed to investigate the impact of nutritional factors, genetics, and exercise on biochemistry and lipoprotein panel analytes in orange-winged Amazon parrots.

  5. A comparative proteomic study of plasma in feline pancreatitis and pancreatic carcinoma using 2-dimensional gel electrophoresis to identify diagnostic biomarkers: A pilot study

    PubMed Central

    Meachem, Melissa D.; Snead, Elisabeth R.; Kidney, Beverly A.; Jackson, Marion L.; Dickinson, Ryan; Larson, Victoria; Simko, Elemir

    2015-01-01

    While pancreatitis is now recognized as a common ailment in cats, the diagnosis remains challenging due to discordant results and suboptimal sensitivity of ultrasound and specific feline pancreatic lipase (Spec fPL) assay. Pancreatitis also shares similar clinical features with pancreatic carcinoma, a rare but aggressive disease with a grave prognosis. The objective of this pilot study was to compare the plasma proteomes of normal healthy cats (n = 6), cats with pancreatitis (n = 6), and cats with pancreatic carcinoma (n = 6) in order to identify potential new biomarkers of feline pancreatic disease. After plasma protein separation by 2-dimensional gel electrophoresis, protein spots were detected by Coomassie Brilliant Blue G-250 staining and identified by mass spectrometry. Alpha-1-acid glycoprotein (AGP), apolipoprotein-A1 (Apo-A1), and apolipoprotein-A1 precursor (Pre Apo-A1) appeared to be differentially expressed, which suggests the presence of a systemic acute-phase response and alteration of lipid metabolism in cats with pancreatic disease. Future studies involving greater case numbers are needed in order to assess the utility of these proteins as potential biomarkers. More sensitive proteomic techniques may also be helpful in detecting significant but low-abundance proteins. PMID:26130850

  6. Copper deficiency in the preterm infant of very low birthweight. Four cases and a reference range for plasma copper.

    PubMed Central

    Sutton, A M; Harvie, A; Cockburn, F; Farquharson, J; Logan, R W

    1985-01-01

    Four preterm infants of very low birthweight (less than 1500 g) developed signs of copper deficiency between age 8 and 10 weeks. All had required prolonged ventilatory support, parenteral nutrition, and nasojejunal feeding. The clinical features, which included osteoporosis, oedema, anaemia, neutropenia, and late apnoea improved when the oral copper intake was increased. Diagnosis was made more difficult because a suitable reference range for plasma copper was not available. Serial measurements of plasma copper in 39 preterm infants who had no important medical problems were used to produce a reference range for plasma copper from 30 weeks' gestation to term plus seven weeks. This information will aid recognition of hypocupraemia in the very low birthweight infant who is particularly at risk of copper deficiency. PMID:4026360

  7. The statistical mapping of magnetosheath plasma properties based on THEMIS measurements in the magnetosheath interplanetary medium reference frame

    NASA Astrophysics Data System (ADS)

    Dimmock, A. P.; Nykyri, K.

    2013-08-01

    The magnetosheath operates as a natural filter between the solar wind and the magnetospheric plasma. As a result of this, the magnetosheath plays a crucial role in the plasma momentum and energy transport from the interplanetary medium into the magnetosphere. Statistical studies of the magnetosheath are difficult due to the dynamic nature of the terrestrial bow shock and the magnetopause. As a result of this, the spatial and temporal dependence of magnetosheath plasma properties under varying solar wind conditions is still not completely understood. We present a study of magnetosheath plasma properties using 5 years of THEMIS and OMNI data to produce statistical maps of fundamental magnetosheath plasma properties. The magnetosheath interplanetary medium reference frame is applied to present data in a normalized reference frame which accounts for both boundary and orbital motion. The statistical maps are compared with the MHD runs from the CCMC-BATS-R-US model which agree favorably. The results are also used to investigate the presence of any magnetosheath plasma parameter asymmetries and their possible causes.

  8. Protein expression profiles of human lymph and plasma mapped by 2D-DIGE and 1D SDS–PAGE coupled with nanoLC–ESI–MS/MS bottom-up proteomics

    PubMed Central

    Clement, Cristina C.; Aphkhazava, David; Nieves, Edward; Callaway, Myrasol; Olszewski, Waldemar; Rotzschke, Olaf; Santambrogio, Laura

    2013-01-01

    In this study a proteomic approach was used to define the protein content of matched samples of afferent prenodal lymph and plasma derived from healthy volunteers. The analysis was performed using two analytical methodologies coupled with nanoliquid chromatography-tandem mass spectrometry: one-dimensional gel electrophoresis (1DEF nanoLC Orbitrap–ESI–MS/MS), and two-dimensional fluorescence difference-in-gel electrophoresis (2D-DIGE nanoLC–ESI–MS/MS). The 253 significantly identified proteins (p<0.05), obtained from the tandem mass spectrometry data, were further analyzed with pathway analysis (IPA) to define the functional signature of prenodal lymph and matched plasma. The 1DEF coupled with nanoLC–MS–MS revealed that the common proteome between the two biological fluids (144 out of 253 proteins) was dominated by complement activation and blood coagulation components, transporters and protease inhibitors. The enriched proteome of human lymph (72 proteins) consisted of products derived from the extracellular matrix, apoptosis and cellular catabolism. In contrast, the enriched proteome of human plasma (37 proteins) consisted of soluble molecules of the coagulation system and cell–cell signaling factors. The functional networks associated with both common and source-distinctive proteomes highlight the principal biological activity of these immunologically relevant body fluids. PMID:23202415

  9. AGT genetic variation, plasma AGT, and blood pressure: An analysis of the Utah Genetic Reference Project pedigrees.

    PubMed

    Watkins, W Scott; Rohrwasser, Andreas; Peiffer, Andy; Leppert, Mark F; Lalouel, Jean-Marc; Jorde, Lynn B

    2010-08-01

    Much remains unknown about the genetic factors that contribute to essential hypertension. The Utah Genetic Reference Project (UGRP) large pedigree collection provides new opportunities to study quantitative relationships between genetic variation, endophenotypes, and blood pressure. We analyzed the relationship between common single-nucleotide polymorphisms (SNPs) and haplotypes spanning the angiotensinogen (AGT) gene and promoter region, plasma AGT levels, and systolic (SBP) and diastolic blood pressure (DBP) in 424 individuals from 41 two-generation UGRP families. Plasma AGT levels are significantly correlated among UGRP family members. Correlations are higher for males than for females. Parent-offspring correlations for plasma AGT (0.30) are higher than those for SBP (0.26) and DBP (0.17) (all P values <0.01). The additive heritability (h(2)) for plasma AGT is high (0.74) and substantially exceeds heritability estimates for SBP (0.26) and DBP (0.16) (all P values <0.01). Significant linkage (logarithm of the odds (LOD) >3) is found between six AGT SNPs and plasma AGT. A model that utilizes three AGT haplotype groups produces the best LOD score (5.1) that exceeds the best single SNP LOD score (3.8). Plasma AGT and blood pressure were not significantly correlated. Plasma AGT levels demonstrate high heritability in 41 UGRP families. Locus-specific heritability estimates for AGT SNPs and haplotypes approach 67%, indicating that variation at AGT accounts for a large percentage of the heritability of plasma AGT. A three-way haplotype model outperforms single SNPs for quantitative linkage analysis to plasma AGT. In these predominantly normotensive individuals, plasma AGT did not correlate significantly with blood pressure.

  10. Sys-BodyFluid: a systematical database for human body fluid proteome research.

    PubMed

    Li, Su-Jun; Peng, Mao; Li, Hong; Liu, Bo-Shu; Wang, Chuan; Wu, Jia-Rui; Li, Yi-Xue; Zeng, Rong

    2009-01-01

    Recently, body fluids have widely become an important target for proteomic research and proteomic study has produced more and more body fluid related protein data. A database is needed to collect and analyze these proteome data. Thus, we developed this web-based body fluid proteome database Sys-BodyFluid. It contains eleven kinds of body fluid proteomes, including plasma/serum, urine, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, synovial fluid, nipple aspirate fluid, tear fluid, seminal fluid, human milk and amniotic fluid. Over 10,000 proteins are presented in the Sys-BodyFluid. Sys-BodyFluid provides the detailed protein annotations, including protein description, Gene Ontology, domain information, protein sequence and involved pathways. These proteome data can be retrieved by using protein name, protein accession number and sequence similarity. In addition, users can query between these different body fluids to get the different proteins identification information. Sys-BodyFluid database can facilitate the body fluid proteomics and disease proteomics research as a reference database. It is available at http://www.biosino.org/bodyfluid/.

  11. Pressurized Pepsin Digestion in Proteomics

    PubMed Central

    López-Ferrer, Daniel; Petritis, Konstantinos; Robinson, Errol W.; Hixson, Kim K.; Tian, Zhixin; Lee, Jung Hwa; Lee, Sang-Won; Tolić, Nikola; Weitz, Karl K.; Belov, Mikhail E.; Smith, Richard D.; Paša-Tolić, Ljiljana

    2011-01-01

    Integrated top-down bottom-up proteomics combined with on-line digestion has great potential to improve the characterization of protein isoforms in biological systems and is amendable to high throughput proteomics experiments. Bottom-up proteomics ultimately provides the peptide sequences derived from the tandem MS analyses of peptides after the proteome has been digested. Top-down proteomics conversely entails the MS analyses of intact proteins for more effective characterization of genetic variations and/or post-translational modifications. Herein, we describe recent efforts toward efficient integration of bottom-up and top-down LC-MS-based proteomics strategies. Since most proteomics separations utilize acidic conditions, we exploited the compatibility of pepsin (where the optimal digestion conditions are at low pH) for integration into bottom-up and top-down proteomics work flows. Pressure-enhanced pepsin digestions were successfully performed and characterized with several standard proteins in either an off-line mode using a Barocycler or an on-line mode using a modified high pressure LC system referred to as a fast on-line digestion system (FOLDS). FOLDS was tested using pepsin and a whole microbial proteome, and the results were compared against traditional trypsin digestions on the same platform. Additionally, FOLDS was integrated with a RePlay configuration to demonstrate an ultrarapid integrated bottom-up top-down proteomics strategy using a standard mixture of proteins and a monkey pox virus proteome. PMID:20627868

  12. The human plasma-metabolome: Reference values in 800 French healthy volunteers; impact of cholesterol, gender and age.

    PubMed

    Trabado, Séverine; Al-Salameh, Abdallah; Croixmarie, Vincent; Masson, Perrine; Corruble, Emmanuelle; Fève, Bruno; Colle, Romain; Ripoll, Laurent; Walther, Bernard; Boursier-Neyret, Claire; Werner, Erwan; Becquemont, Laurent; Chanson, Philippe

    2017-01-01

    Metabolomic approaches are increasingly used to identify new disease biomarkers, yet normal values of many plasma metabolites remain poorly defined. The aim of this study was to define the "normal" metabolome in healthy volunteers. We included 800 French volunteers aged between 18 and 86, equally distributed according to sex, free of any medication and considered healthy on the basis of their medical history, clinical examination and standard laboratory tests. We quantified 185 plasma metabolites, including amino acids, biogenic amines, acylcarnitines, phosphatidylcholines, sphingomyelins and hexose, using tandem mass spectrometry with the Biocrates AbsoluteIDQ p180 kit. Principal components analysis was applied to identify the main factors responsible for metabolome variability and orthogonal projection to latent structures analysis was employed to confirm the observed patterns and identify pattern-related metabolites. We established a plasma metabolite reference dataset for 144/185 metabolites. Total blood cholesterol, gender and age were identified as the principal factors explaining metabolome variability. High total blood cholesterol levels were associated with higher plasma sphingomyelins and phosphatidylcholines concentrations. Compared to women, men had higher concentrations of creatinine, branched-chain amino acids and lysophosphatidylcholines, and lower concentrations of sphingomyelins and phosphatidylcholines. Elderly healthy subjects had higher sphingomyelins and phosphatidylcholines plasma levels than young subjects. We established reference human metabolome values in a large and well-defined population of French healthy volunteers. This study provides an essential baseline for defining the "normal" metabolome and its main sources of variation.

  13. The human plasma-metabolome: Reference values in 800 French healthy volunteers; impact of cholesterol, gender and age

    PubMed Central

    Al-Salameh, Abdallah; Croixmarie, Vincent; Masson, Perrine; Corruble, Emmanuelle; Fève, Bruno; Colle, Romain; Ripoll, Laurent; Walther, Bernard; Boursier-Neyret, Claire; Werner, Erwan; Becquemont, Laurent; Chanson, Philippe

    2017-01-01

    Metabolomic approaches are increasingly used to identify new disease biomarkers, yet normal values of many plasma metabolites remain poorly defined. The aim of this study was to define the “normal” metabolome in healthy volunteers. We included 800 French volunteers aged between 18 and 86, equally distributed according to sex, free of any medication and considered healthy on the basis of their medical history, clinical examination and standard laboratory tests. We quantified 185 plasma metabolites, including amino acids, biogenic amines, acylcarnitines, phosphatidylcholines, sphingomyelins and hexose, using tandem mass spectrometry with the Biocrates AbsoluteIDQ p180 kit. Principal components analysis was applied to identify the main factors responsible for metabolome variability and orthogonal projection to latent structures analysis was employed to confirm the observed patterns and identify pattern-related metabolites. We established a plasma metabolite reference dataset for 144/185 metabolites. Total blood cholesterol, gender and age were identified as the principal factors explaining metabolome variability. High total blood cholesterol levels were associated with higher plasma sphingomyelins and phosphatidylcholines concentrations. Compared to women, men had higher concentrations of creatinine, branched-chain amino acids and lysophosphatidylcholines, and lower concentrations of sphingomyelins and phosphatidylcholines. Elderly healthy subjects had higher sphingomyelins and phosphatidylcholines plasma levels than young subjects. We established reference human metabolome values in a large and well-defined population of French healthy volunteers. This study provides an essential baseline for defining the “normal” metabolome and its main sources of variation. PMID:28278231

  14. Establishment of reference intervals for plasma protein electrophoresis in Indo-Pacific green sea turtles, Chelonia mydas.

    PubMed

    Flint, Mark; Matthews, Beren J; Limpus, Colin J; Mills, Paul C

    2015-01-01

    Biochemical and haematological parameters are increasingly used to diagnose disease in green sea turtles. Specific clinical pathology tools, such as plasma protein electrophoresis analysis, are now being used more frequently to improve our ability to diagnose disease in the live animal. Plasma protein reference intervals were calculated from 55 clinically healthy green sea turtles using pulsed field electrophoresis to determine pre-albumin, albumin, α-, β- and γ-globulin concentrations. The estimated reference intervals were then compared with data profiles from clinically unhealthy turtles admitted to a local wildlife hospital to assess the validity of the derived intervals and identify the clinically useful plasma protein fractions. Eighty-six per cent {19 of 22 [95% confidence interval (CI) 65-97]} of clinically unhealthy turtles had values outside the derived reference intervals, including the following: total protein [six of 22 turtles or 27% (95% CI 11-50%)], pre-albumin [two of five, 40% (95% CI 5-85%)], albumin [13 of 22, 59% (95% CI 36-79%)], total albumin [13 of 22, 59% (95% CI 36-79%)], α- [10 of 22, 45% (95% CI 24-68%)], β- [two of 10, 20% (95% CI 3-56%)], γ- [one of 10, 10% (95% CI 0.3-45%)] and β-γ-globulin [one of 12, 8% (95% CI 0.2-38%)] and total globulin [five of 22, 23% (8-45%)]. Plasma protein electrophoresis shows promise as an accurate adjunct tool to identify a disease state in marine turtles. This study presents the first reference interval for plasma protein electrophoresis in the Indo-Pacific green sea turtle.

  15. Establishment of reference intervals for plasma protein electrophoresis in Indo-Pacific green sea turtles, Chelonia mydas

    PubMed Central

    Flint, Mark; Matthews, Beren J.; Limpus, Colin J.; Mills, Paul C.

    2015-01-01

    Biochemical and haematological parameters are increasingly used to diagnose disease in green sea turtles. Specific clinical pathology tools, such as plasma protein electrophoresis analysis, are now being used more frequently to improve our ability to diagnose disease in the live animal. Plasma protein reference intervals were calculated from 55 clinically healthy green sea turtles using pulsed field electrophoresis to determine pre-albumin, albumin, α-, β- and γ-globulin concentrations. The estimated reference intervals were then compared with data profiles from clinically unhealthy turtles admitted to a local wildlife hospital to assess the validity of the derived intervals and identify the clinically useful plasma protein fractions. Eighty-six per cent {19 of 22 [95% confidence interval (CI) 65–97]} of clinically unhealthy turtles had values outside the derived reference intervals, including the following: total protein [six of 22 turtles or 27% (95% CI 11–50%)], pre-albumin [two of five, 40% (95% CI 5–85%)], albumin [13 of 22, 59% (95% CI 36–79%)], total albumin [13 of 22, 59% (95% CI 36–79%)], α- [10 of 22, 45% (95% CI 24–68%)], β- [two of 10, 20% (95% CI 3–56%)], γ- [one of 10, 10% (95% CI 0.3–45%)] and β–γ-globulin [one of 12, 8% (95% CI 0.2–38%)] and total globulin [five of 22, 23% (8–45%)]. Plasma protein electrophoresis shows promise as an accurate adjunct tool to identify a disease state in marine turtles. This study presents the first reference interval for plasma protein electrophoresis in the Indo-Pacific green sea turtle. PMID:27293722

  16. SPM analysis of parametric (R)-[11C]PK11195 binding images: plasma input versus reference tissue parametric methods.

    PubMed

    Schuitemaker, Alie; van Berckel, Bart N M; Kropholler, Marc A; Veltman, Dick J; Scheltens, Philip; Jonker, Cees; Lammertsma, Adriaan A; Boellaard, Ronald

    2007-05-01

    (R)-[11C]PK11195 has been used for quantifying cerebral microglial activation in vivo. In previous studies, both plasma input and reference tissue methods have been used, usually in combination with a region of interest (ROI) approach. Definition of ROIs, however, can be labourious and prone to interobserver variation. In addition, results are only obtained for predefined areas and (unexpected) signals in undefined areas may be missed. On the other hand, standard pharmacokinetic models are too sensitive to noise to calculate (R)-[11C]PK11195 binding on a voxel-by-voxel basis. Linearised versions of both plasma input and reference tissue models have been described, and these are more suitable for parametric imaging. The purpose of this study was to compare the performance of these plasma input and reference tissue parametric methods on the outcome of statistical parametric mapping (SPM) analysis of (R)-[11C]PK11195 binding. Dynamic (R)-[11C]PK11195 PET scans with arterial blood sampling were performed in 7 younger and 11 elderly healthy subjects. Parametric images of volume of distribution (Vd) and binding potential (BP) were generated using linearised versions of plasma input (Logan) and reference tissue (Reference Parametric Mapping) models. Images were compared at the group level using SPM with a two-sample t-test per voxel, both with and without proportional scaling. Parametric BP images without scaling provided the most sensitive framework for determining differences in (R)-[11C]PK11195 binding between younger and elderly subjects. Vd images could only demonstrate differences in (R)-[11C]PK11195 binding when analysed with proportional scaling due to intersubject variation in K1/k2 (blood-brain barrier transport and non-specific binding).

  17. Pediatric reference intervals for plasma free and total metanephrines established with a parametric approach: relevance to the diagnosis of neuroblastoma.

    PubMed

    Franscini, Laura Crosazzo; Vazquez-Montes, Maria; Buclin, Thierry; Perera, Rafael; Dunand, Marielle; Grouzmann, Eric; Beck-Popovic, Maja

    2015-04-01

    Urine catecholamines, vanillylmandelic, and homovanillic acid are recognized biomarkers for the diagnosis and follow-up of neuroblastoma. Plasma free (f) and total (t) normetanephrine (NMN), metanephrine (MN) and methoxytyramine (MT) could represent a convenient alternative to those urine markers. The primary objective of this study was to establish pediatric centile charts for plasma metanephrines. Secondarily, we explored their diagnostic performance in 10 patients with neuroblastoma. We recruited 191 children (69 females) free of neuroendocrine disease to establish reference intervals for plasma metanephrines, reported as centile curves for a given age and sex based on a parametric method using fractional polynomials models. Urine markers and plasma metanephrines were measured in 10 children with neuroblastoma at diagnosis. Plasma total metanephrines were measured by HPLC with coulometric detection and plasma free metanephrines by tandem LC-MS. We observed a significant age-dependence for tNMN, fNMN, and fMN, and a gender and age-dependence for tMN, fNMN, and fMN. Free MT was below the lower limit of quantification in 94% of the children. All patients with neuroblastoma at diagnosis were above the 97.5th percentile for tMT, tNMN, fNMN, and fMT, whereas their fMN and tMN were mostly within the normal range. As expected, urine assays were inconstantly predictive of the disease. A continuous model incorporating all data for a given analyte represents an appealing alternative to arbitrary partitioning of reference intervals across age categories. Plasma metanephrines are promising biomarkers for neuroblastoma, and their performances need to be confirmed in a prospective study on a large cohort of patients. © 2015 Wiley Periodicals, Inc.

  18. Efficient isolation and proteomic analysis of cell plasma membrane proteins in gastric cancer reveal a novel differentiation and progression related cell surface marker, R-cadherin.

    PubMed

    Chen, Bo; Luo, Qi-Cong; Chen, Jian-Bo; Lin, Li-E; Luo, Ming-Xu; Ren, Hong-Yue; Chen, Pei-Qiong; Shi, Lian-Guo

    2016-09-01

    Cell plasma membrane proteins, playing a crucial role in cell malignant transformation and development, were the main targets of tumor detection and therapy. In this study, CyDye/biotin double-labeling proteomic approach was adopted to profile the membrane proteome of gastric cancer cell line BGC-823 and paired immortalized gastric epithelial cell GES-1. Real-time PCR, Western blotting, and immunohistochemical staining were used to validate the differential expression of a novel identified cell surface marker R-cadherin in gastric cancer cells and tissues. Clinicopathological study and survival analysis were performed to estimate its roles in tumor progression and outcome prediction. Real-time PCR and Western blotting showed that the expression level of R-cadherin in gastric cancer were significantly lower than non-cancerous epithelial cell and tissues. Clinicopathological study indicated that R-cadherin was dominantly expressed on cell surface of normal gastric epithelium, and its expression deletion in gastric cancer tissues was associated with tumor site, differentiation, lymph node metastasis, and pTNM (chi-square test, P < 0.05). Those patients with R-cadherin positive expression displayed better overall survivals than negative expression group (log-rank test, P = 0.000). Cox multivariate survival analysis revealed lacking the expression of R-cadherin was a main independent predictor for poor clinical outcome in gastric cancer (RR = 5.680, 95 % CI 2.250-14.341, P < 0.01). We have established a fundamental membrane proteome database for gastric cancer and identified R-cadherin as a tumor differentiation and progression-related cell surface marker of gastric cancer. Lacking the expression of R-cadherin indicates poor prognosis in patients with gastric cancer.

  19. In between - Proteomics of dog biological fluids.

    PubMed

    Miller, Ingrid; Preßlmayer-Hartler, Andrea; Wait, Robin; Hummel, Karin; Sensi, Cristina; Eberini, Ivano; Razzazi-Fazeli, Ebrahim; Gianazza, Elisabetta

    2014-06-25

    Dogs are relevant to biomedical research in connection both to veterinary medicine for their role as pets and to basic investigations for their use as animal models in pathology, pharmacology and toxicology studies. Proteomic analysis of biological fluids is less advanced for dogs than for other animal species but a wealth of information has already been gathered, which we summarize in this review. As a remarkable feature, we also assemble here for due reference a number of 2-DE serum/plasma or urine patterns in health and disease; some of them correspond to unpublished data from the University of Veterinary Medicine Vienna. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Use of Differential Scanning Calorimetry and Immunoaffinity Chromatography to Identify Disease Induced Changes in Human Blood Plasma Proteome.

    PubMed

    Brudar, Sandi; Černigoj, Urh; Podgornik, Helena; Kržan, Mojca; Prislan, Iztok

    2017-09-01

    Differential scanning calorimetry provides unique signatures of blood plasma samples. Plasma samples from diseased individuals yield specific thermograms, which differ from each other and from plasma samples of healthy individuals. Thermograms from individuals suffering from chronic lymphocytic leukemia, multiple myeloma and acute myeloid leukemia were measured with DSC. To obtain additional information about thermal behaviour of plasma proteins immunoaffinity chromatography was introduced. An immunoextraction of HSA using a chromatographic column with immobilized anti-HSA was carried out in order to enrich less abundant plasma proteins, which could provide a further insight into disease development. Efficiency of HSA depletion and protein composition of fractionated plasma was validated by SDS-PAGE.

  1. Identification of a Novel Function of Adipocyte Plasma Membrane-Associated Protein (APMAP) in Gestational Diabetes Mellitus by Proteomic Analysis of Omental Adipose Tissue.

    PubMed

    Ma, Yuhang; Gao, Jing; Yin, Jiajing; Gu, Liping; Liu, Xing; Chen, Su; Huang, Qianfang; Lu, Huifang; Yang, Yuemin; Zhou, Hu; Wang, Yufan; Peng, Yongde

    2016-02-05

    Gestational diabetes mellitus (GDM) is considered as an early stage of type 2 diabetes mellitus. In this study, we compared demographic and clinical data between six GDM subjects and six normal glucose tolerance (NGT; healthy controls) subjects and found that homeostasis model of assessment for insulin resistance index (HOMA-IR) increased in GDM. Many previous studies demonstrated that omental adipose tissue dysfunction could induce insulin resistance. Thus, to investigate the cause of insulin resistance in GDM, we used label-free proteomics to identify differentially expressed proteins in omental adipose tissues from GDM and NGT subjects (data are available via ProteomeXchange with identifier PXD003095). A total of 3528 proteins were identified, including 66 significantly changed proteins. Adipocyte plasma membrane-associated protein (APMAP, a.k.a. C20orf3), one of the differentially expressed proteins, was down-regulated in GDM omental adipose tissues. Furthermore, mature 3T3-L1 adipocytes were used to simulate omental adipocytes. The inhibition of APMAP expression by RNAi impaired insulin signaling and activated NFκB signaling in these adipocytes. Our study revealed that the down-regulation of APMAP in omental adipose tissue may play an important role in insulin resistance in the pathophysiology of GDM.

  2. Effect of the reference electrode size on the ionization instability in the plasma sheath of a small positively biased electrode

    SciTech Connect

    Bliokh, Y. P.; Brodsky, Yu. L.; Chashka, Kh. B.; Felsteiner, J.; Slutsker, Ya. Z.

    2011-06-01

    It is well known that additional ionization in the vicinity of a positively biased electrode immersed into a weakly ionized plasma is responsible for a hysteresis in the electrode current-voltage characteristics and the current self-oscillations rise. Here we show both experimentally and theoretically that under certain conditions these phenomena cannot be correctly interpreted once considered separately from the reference electrode current-voltage characteristics. It is shown that small electrodes can be separated into three groups according to the relation between the electrode and the reference electrode areas. Each group is characterized by its own dependence of the collected current on the bias voltage.

  3. Proteome changes in the plasma of Papilio xuthus (Lepidoptera: Papilionidae): effect of parasitization by the endoparasitic wasp Pteromalus puparum (Hymenoptera: Pteromalidae).

    PubMed

    Zhu, Jia-ying; Ye, Gong-yin; Fang, Qi; Hu, Cui

    2009-06-01

    Although the biochemical dissection of parasitoid-host interactions is becoming well characterized, the molecular knowledge concerning them is minimal. In order to understand the molecular bases of the host immune response to parasitoid attack, we explored the response of Papilio xuthus parasitized by the endoparasitic wasp Pteromalus puparum using proteomic approach. By examining the differential expression of plasma proteins in the parasitized and unparasitized host pupae by two-dimensional (2D) electrophoresis, 16 proteins were found to vary in relation to parasitization compared with unparasitized control samples. All of them were submitted to identification by mass spectrometry coupled with a database search. The modulated proteins were found to fall into the following functional groups: humoral or cellular immunity, detoxification, energy metabolism, and others. This study contributes insights into the molecular mechanism of the relationships between parasitoids and their host insects.

  4. Clinical studies on plasma fibronectin and factor XIII; with special reference to hyperlipoproteinemia.

    PubMed

    Cucuianu, M; Rus, H G; Cristea, A; Niculescu, F; Bedeleanu, D; Poruţiu, D; Roman, S

    1985-04-30

    When compared to age-matched normal weight normolipidemic control subjects, plasma factor XIII, plasma fibronectin and serum cholinesterase levels were found to be markedly decreased in patients with decompensated cirrhosis of the liver, not significantly changed in hyperlipoproteinemia type IIa (heterozygous subjects) and increased in hypertriglyceridemic subjects (type IIb and IV) as well as in hyperlipidemic nephrotic patients. A possible accelerated hepatic synthesis of certain plasma proteins including factor XIII and fibronectin in patients with the nephrotic syndrome as well as in endogenous hypertriglyceridemia is envisaged. It is also considered that mural thrombi, richer in factor XIII and fibronectin, would be more resistant to fibrinolysis and more readily attached to subendothelial structures.

  5. Evaluation of plasma proteomic data for Alzheimer disease state classification and for the prediction of progression from mild cognitive impairment to Alzheimer disease.

    PubMed

    Llano, Daniel A; Devanarayan, Viswanath; Simon, Adam J

    2013-01-01

    Previous studies that have examined the potential for plasma markers to serve as biomarkers for Alzheimer disease (AD) have studied single analytes and focused on the amyloid-β and τ isoforms and have failed to yield conclusive results. In this study, we performed a multivariate analysis of 146 plasma analytes (the Human DiscoveryMAP v 1.0 from Rules-Based Medicine) in 527 subjects with AD, mild cognitive impairment (MCI), or cognitively normal elderly subjects from the Alzheimer's Disease Neuroimaging Initiative database. We identified 4 different proteomic signatures, each using 5 to 14 analytes, that differentiate AD from control patients with sensitivity and specificity ranging from 74% to 85%. Five analytes were common to all 4 signatures: apolipoprotein A-II, apolipoprotein E, serum glutamic oxaloacetic transaminase, α-1-microglobulin, and brain natriuretic peptide. None of the signatures adequately predicted progression from MCI to AD over a 12- and 24-month period. A new panel of analytes, optimized to predict MCI to AD conversion, was able to provide 55% to 60% predictive accuracy. These data suggest that a simple panel of plasma analytes may provide an adjunctive tool to differentiate AD from controls, may provide mechanistic insights to the etiology of AD, but cannot adequately predict MCI to AD conversion.

  6. Proteomics analysis of vesicles isolated from plasma and urine of prostate cancer patients using a multiplex, aptamer-based protein array.

    PubMed

    Welton, Joanne Louise; Brennan, Paul; Gurney, Mark; Webber, Jason Paul; Spary, Lisa Kate; Carton, David Gil; Falcón-Pérez, Juan Manuel; Walton, Sean Peter; Mason, Malcolm David; Tabi, Zsuzsanna; Clayton, Aled

    2016-01-01

    Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasive disease markers. Obtaining vesicles of sufficient quality and quantity for profiling studies has, however, been a major problem, as samples are often replete with co-isolated material that can interfere with the identification of genuine low abundance, vesicle components. Here, we used a combination of ultracentrifugation and size-exclusion chromatography to isolate and analyse vesicles of plasma or urine origin. We describe a sample-handling workflow that gives reproducible, quality vesicle isolations sufficient for subsequent protein profiling. Using a semi-quantitative aptamer-based protein array, we identified around 1,000 proteins, of which almost 400 were present at comparable quantities in plasma versus urine vesicles. Significant differences were, however, apparent with elements like HSP90, integrin αVβ5 and Contactin-1 more prevalent in urinary vesicles, while hepatocyte growth factor activator, prostate-specific antigen-antichymotrypsin complex and many others were more abundant in plasma vesicles. This was also applied to a small set of specimens collected from men with metastatic prostate cancer, highlighting several proteins with the potential to indicate treatment refractory disease. The study provides a practical platform for furthering protein profiling of vesicles in prostate cancer, and, hopefully, many other disease scenarios.

  7. Proteomics analysis of vesicles isolated from plasma and urine of prostate cancer patients using a multiplex, aptamer-based protein array

    PubMed Central

    Welton, Joanne Louise; Brennan, Paul; Gurney, Mark; Webber, Jason Paul; Spary, Lisa Kate; Carton, David Gil; Falcón-Pérez, Juan Manuel; Walton, Sean Peter; Mason, Malcolm David; Tabi, Zsuzsanna; Clayton, Aled

    2016-01-01

    Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasive disease markers. Obtaining vesicles of sufficient quality and quantity for profiling studies has, however, been a major problem, as samples are often replete with co-isolated material that can interfere with the identification of genuine low abundance, vesicle components. Here, we used a combination of ultracentrifugation and size-exclusion chromatography to isolate and analyse vesicles of plasma or urine origin. We describe a sample-handling workflow that gives reproducible, quality vesicle isolations sufficient for subsequent protein profiling. Using a semi-quantitative aptamer-based protein array, we identified around 1,000 proteins, of which almost 400 were present at comparable quantities in plasma versus urine vesicles. Significant differences were, however, apparent with elements like HSP90, integrin αVβ5 and Contactin-1 more prevalent in urinary vesicles, while hepatocyte growth factor activator, prostate-specific antigen–antichymotrypsin complex and many others were more abundant in plasma vesicles. This was also applied to a small set of specimens collected from men with metastatic prostate cancer, highlighting several proteins with the potential to indicate treatment refractory disease. The study provides a practical platform for furthering protein profiling of vesicles in prostate cancer, and, hopefully, many other disease scenarios. PMID:27363484

  8. Assessments of feline plasma biochemistry reference intervals for three in-house analysers and a commercial laboratory analyser.

    PubMed

    Baral, Randolph M; Dhand, Navneet K; Krockenberger, Mark B; Govendir, Merran

    2015-08-01

    For each species, the manufacturers of in-house analysers (and commercial laboratories) provide standard reference intervals (RIs) that do not account for any differences such as geographical population differences and do not overtly state the potential for variation between results obtained from serum or plasma. Additionally, biases have been demonstrated for in-house analysers which result in different RIs for each different type of analyser. The objective of this study was to calculate RIs (with 90% confidence intervals [CIs]) for 13 biochemistry analytes when tested on three commonly used in-house veterinary analysers, as well as a commercial laboratory analyser. The calculated RIs were then compared with those provided by the in-house analyser manufacturers and the commercial laboratory. Plasma samples were collected from 53 clinically normal cats. After centrifugation, plasma was divided into four aliquots; one aliquot was sent to the commercial laboratory and the remaining three were tested using the in-house biochemistry analysers. The distribution of results was used to choose the appropriate statistical technique for each analyte from each analyser to calculate RIs. Provided reference limits were deemed appropriate if they fell within the 90% CIs of the calculated reference limits. Transference validation was performed on provided and calculated RIs. Twenty-nine of a possible 102 provided reference limits (28%) were within the calculated 90% CIs. To ensure proper interpretation of laboratory results, practitioners should determine RIs for their practice populations and/or use reference change values when assessing their patients' clinical chemistry results. © ISFM and AAFP 2014.

  9. Reference intervals for plasma biochemical and hematologic measures in loggerhead sea turtles (Caretta caretta) from Moreton Bay, Australia.

    PubMed

    Flint, Mark; Morton, John M; Limpus, Colin J; Patterson-Kane, Janet C; Mills, Paul C

    2010-07-01

    Biochemical and hematologic reference intervals have been reported for loggerhead sea turtles (Caretta caretta, Linnaeus 1758), but low sample numbers and simple statistical analyses have constrained their diagnostic usefulness. During June 2007-May 2008, 101 loggerhead sea turtles in Moreton Bay, Queensland, Australia, were captured by hand from boats; clinically assessed to determine health status; blood was sampled; and biochemical and hematologic variables were measured. Of these turtles, 66 were classified as clinically healthy and 23 as unhealthy. Reference intervals were calculated using data from clinically healthy turtles. Of the clinically unhealthy turtles, 82 and 45% had at least one biochemical and hematologic result, respectively, outside of at least one of the calculated intervals. However, only low proportions of unhealthy loggerhead sea turtles had abnormal results for each variable. The highest percentage of unhealthy turtles that were outside at least one estimated reference interval was 35%, for thrombocyte counts. Neither sex nor maturity category (mature versus large immature) influenced the risk of being clinically unhealthy. These are the first plasma biochemical and hematologic reference intervals reported for loggerhead sea turtles from the southwestern Pacific Ocean. We conclude that, for loggerhead sea turtles in Moreton Bay, separate reference intervals are required for mature and immature turtles for thrombocyte counts and for male and female turtles for lymphocyte, heterophil, and total white cell counts; otherwise, a single reference interval can be used regardless of age or sex. When estimating reference intervals in loggerhead sea turtles, it is desirable to use both methods for calculating reference intervals used in this study because intervals can differ substantially between methods for some variables. Joint interpretation using reference intervals from both methods allows the categorization of results as "normal," "suspect

  10. Physics and applications of atmospheric non-thermal air plasma with reference to environment

    NASA Astrophysics Data System (ADS)

    Marode, E.; Djermoune, D.; Dessante, P.; Deniset, C.; Ségur, P.; Bastien, F.; Bourdon, A.; Laux, C.

    2009-12-01

    Since air is a natural part of our environment, special attention is given to the study of plasmas in air at atmospheric pressure and their applications. This fact promoted the study of electrical conduction in air-like mixtures, i.e. mixtures containing an electronegative gas component. If the ionization growth is not limited its temporal evolution leads to spark formation, i.e. a thermal plasma of several thousand kelvins in a quasi-local thermodynamic equilibrium state. But before reaching such a thermal state, a plasma sets up where the electrons increase their energy characterized by an electron temperature Te much higher than that of heavy species T or T+ for the ions. Since the plasma is no longer characterized by only one temperature T, it is said to be in a non-thermal plasma (NTP) state. Practical ways are listed to prevent electron ionization from going beyond the NTP states. Much understanding of such NTP may be gathered from the study of the simple paradigmatic case of a discharge induced between a sharp positively stressed point electrode facing a grounded negative plane electrode. Some physical properties will be gathered from such configurations and links underlined between these properties and some associated applications, mostly environmental. Aerosol filtration and electrostatic precipitators, pollution control by removal of hazardous species contained in flue gas exhaust, sterilization applications for medical purposes and triggering fuel combustion in vehicle motors are among such applications nowadays.

  11. Plasma vitamin B12 concentrations and the risk of colorectal cancer: a nested case-referent study.

    PubMed

    Dahlin, Anna M; Van Guelpen, Bethany; Hultdin, Johan; Johansson, Ingegerd; Hallmans, Göran; Palmqvist, Richard

    2008-05-01

    In this nested case-referent study, we related plasma concentrations of vitamin B12 to the risk of colorectal cancer, taking into consideration prediagnostic plasma folate and total homocysteine concentrations. Subjects were 226 cases and double matched referents from the population-based Northern Sweden Health and Disease Study. Follow-up times from recruitment to diagnosis ranged from 0.1 to 12.7 years, with a median of 4.2 years. Plasma vitamin B12 concentrations were inversely associated with the risk of rectal cancer: univariate odds ratio for the highest versus lowest quintile 0.34 (95% confidence interval (95% CI) 0.13-0.83), p(trend) = 0.004. Risk estimates were attenuated slightly but remained statistically significant after adjustment for body mass index, current smoking, recreational and occupational physical activity, alcohol intake and prediagnostic plasma folate and total homocysteine concentrations: OR 0.30 (95% CI 0.08-0.99), p(trend) = 0.025. The corresponding univariate and fully adjusted odds ratios for colon cancer were 1.25 (CI 0.66-2.36), p(trend) = 0.185 and 1.42 (CI 0.67-3.05), p(trend) = 0.113, respectively. The observed over-risk was attributable to left-sided colon cancer. Interaction analyses including vitamin B12, folate and homocysteine were in line with the results for vitamin B12 alone. In conclusion, these results suggest that increasing levels of plasma vitamin B12, alone or together with other factors involved in one-carbon metabolism, may reduce the risk of rectal cancer, whereas for colon cancer, the association appears to be less clear. (c) 2007 Wiley-Liss, Inc.

  12. Reference values for hematology and plasma biochemistry variables, and protein electrophoresis of healthy Hermann's tortoises (Testudo hermanni ssp.).

    PubMed

    Andreani, Giulia; Carpenè, Emilio; Cannavacciuolo, Annunziata; Di Girolamo, Nicola; Ferlizza, Enea; Isani, Gloria

    2014-12-01

    Hermann's tortoise, Testudo hermanni, is currently on the International Union for Conservation of Nature (IUCN) red list of endangered species. Reptile medicine relies also on laboratory analyses to evaluate health status, but reference ranges for hematology and biochemistry variables and protein electrophoresis in plasma of healthy tortoises are not available. The purposes of this study were to establish reference ranges for select hematologic and biochemical variables in clinically healthy Hermann's tortoises, and evaluate the impact of sex and season. Blood samples were collected from 34 healthy tortoises at the end of September and beginning of July. Blood smears, HCT, concentrations of HGB and select plasma biochemical analytes, select enzyme activities, and plasma protein fractions were evaluated. Reference ranges were determined and checked for influence of sex and sampling time point. Typical reptilian RBC and WBC were observed in blood smears. HCT and concentrations of HGB, uric acid and urea, and ALT and AST activities were significantly higher in males than in females. Concentrations of glucose, uric acid, and phosphate, and AST activity were significantly higher at the beginning of July, whereas concentrations of urea and Cl were higher at the end of September prior to hibernation. The electrophoretic protein fractions included albumin, and α, β, and γ globulins. The reference ranges defined in the present study are useful for clinical tortoise medicine and conservation. Sex and seasonal sampling were identified as factors significantly affecting hematology and blood chemistry analytes; they should be taken into consideration when assessing tortoise health status. © 2014 American Society for Veterinary Clinical Pathology.

  13. Hematologic and plasma biochemical reference values in Indian peafowl (Pavo cristatus).

    PubMed

    Samour, Jaime; Naldo, Jesus; Rahman, Habeeb; Sakkir, Mohammed

    2010-06-01

    Blood samples were collected from captive, adult, clinically normal Indian peafowl (Pavo cristatus) for hematologic and plasma biochemical analyses. Hematologic parameters investigated were total red blood cell count, hemoglobin, packed cell volume, fibrinogen, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, total white blood cell count, differential white blood cell count, and thrombocyte count. Plasma biochemical parameters investigated were alanine aminotransferase, alkaline phosphatase, amylase, aspartate aminotransferase, bile acids, total bilirubin, blood urea nitrogen, calcium, cholesterol, creatinine, creatine kinase, gamma glutamyltransferase, lactate dehydrogenase, glucose, iron, phosphorus, and uric acid, as well as plasma protein electrophoresis. Results were compared with values from studies done in houbara bustards (Chlamydotis undulata), kori bustards (Ardeotis kori), stone curlews (Burhinus oedicnemus), and taxonomically related species, including ring-necked pheasants (Phasianus colchicus), red-legged partridges (Alectoris rufa), Kashmir native fowl (Kashmirfavorella), and Bangladesh native, Fayoumi, and Assil fowl (Gallus domesticus).

  14. Comparative Studies of the Proteome, Glycoproteome, and N-Glycome of Clear Cell Renal Cell Carcinoma Plasma before and after Curative Nephrectomy

    PubMed Central

    2015-01-01

    Clear cell renal cell carcinoma is the most prevalent of all reported kidney cancer cases, and currently there are no markers for early diagnosis. This has stimulated great research interest recently because early detection of the disease can significantly improve the low survival rate. Combining the proteome, glycoproteome, and N-glycome data from clear cell renal cell carcinoma plasma has the potential of identifying candidate markers for early diagnosis and prognosis and/or to monitor disease recurrence. Here, we report on the utilization of a multi-dimensional fractionation approach (12P-M-LAC) and LC–MS/MS to comprehensively investigate clear cell renal cell carcinoma plasma collected before (disease) and after (non-disease) curative nephrectomy (n = 40). Proteins detected in the subproteomes were investigated via label-free quantification. Protein abundance analysis revealed a number of low-level proteins with significant differential expression levels in disease samples, including HSPG2, CD146, ECM1, SELL, SYNE1, and VCAM1. Importantly, we observed a strong correlation between differentially expressed proteins and clinical status of the patient. Investigation of the glycoproteome returned 13 candidate glycoproteins with significant differential M-LAC column binding. Qualitative analysis indicated that 62% of selected candidate glycoproteins showed higher levels (upregulation) in M-LAC bound fraction of disease samples. This observation was further confirmed by released N-glycans data in which 53% of identified N-glycans were present at different levels in plasma in the disease vs non-disease samples. This striking result demonstrates the potential for significant protein glycosylation alterations in clear cell renal cell carcinoma cancer plasma. With future validation in a larger cohort, information derived from this study may lead to the development of clear cell renal cell carcinoma candidate biomarkers. PMID:25184692

  15. Plasma biochemical reference values in clinically healthy captive bearded dragons (Pogona vitticeps) and the effects of sex and season.

    PubMed

    Tamukai, Kenichi; Takami, Yoshinori; Akabane, Yoshihito; Kanazawa, Yuko; Une, Yumi

    2011-09-01

    Bearded dragons are one of the most popular pet lizard species, and biochemical reference values are useful for health management of these reptiles. The objectives of this study were to measure plasma biochemical values in healthy captive bearded dragons, determine reference values, and evaluate the effects of sex and season on the results. Blood samples were collected from 100 captive healthy bearded dragons in Tokyo during the summer and winter. Plasma biochemical measurements were performed using a dry-slide automated biochemical analyzer. The data were then compared based on sex and season using 2-way ANOVA. Globulin, cholesterol, and calcium concentrations of females were higher in both summer and winter compared with the values obtained for males. Both males and females had higher uric acid concentrations in winter than in summer. When compared with males, females had a higher chloride concentration in summer and a higher total protein concentration and aspartate aminotransferase activity in winter. Potassium concentration in males was lower in winter than in summer, whereas in females cholesterol concentration was lower in winter than in summer. Biochemical values that differed based on sex and season in bearded dragons were similar to those in other lizards. These differences reflect physiologic differences in reproductive status in females and seasonal changes in temperature and hydration status. Plasma biochemical values established for bearded dragons in this study will be useful in the diagnostic assessment of captive animals. ©2011 American Society for Veterinary Clinical Pathology.

  16. Plasma butyrylcholinesterase concentrations in psittacine birds: reference values, factors of variation, and association with feather-damaging behavior.

    PubMed

    Grosset, Claire; Bougerol, Christian; Sanchez-Migallon Guzman, David

    2014-03-01

    Butyrylcholinesterase is a glycoprotein enzyme used in the diagnosis of toxicosis by cholinesterase-inhibitor agents like organophosphates and carbamates. In animals, butyrylcholinesterase concentrations have been shown to vary depending on numerous factors such as age, sex, diet, and season of sampling. To establish reference values of plasma butyrylcholinesterase concentrations in common psittacine species, plasma butyrylcholinesterase concentrations were measured in 1942 companion psittacine birds. The birds were classified by age, sex, season, health status, and the presence of feather-damaging behavior. A significant difference was observed among species, with eclectus parrots (Eclectus roratus) having the lowest and African grey parrots (Psittacus erithacus) having the highest reference values. Plasma butyrylcholinesterase concentrations varied by age, health status, and season but not by sex. Concentrations were significantly higher during autumn and spring than during winter and summer, and significantly lower in healthy birds than in sick birds. No significant association between butyrylcholinesterase concentrations and feather-damaging behavior could be established except in lovebirds (Agapornis species). Further research is needed to better understand the effect of nutritional and hormonal factors on butyrylcholinesterase concentrations in psittacine birds and its possible effect on bird cognition.

  17. Microparticle content of plasma for transfusion is influenced by the whole blood hold conditions: pre-analytical considerations for proteomic investigations

    PubMed Central

    Sparrow, Rosemary L.; Sze-Kei Chan, Kasey

    2014-01-01

    Microparticles (MPs) are shed from normal blood cells and may contribute to the coagulation potential of plasma. Transfusion of fresh frozen plasma (FFP) is used to correct coagulopathies and blood loss in trauma or major surgery. The role of MPs in FFP clinical efficacy is unknown. Regulations that govern the preparation of FFP vary in different countries. The aim of this study was to determine the effect of whole blood (WB)-hold conditions before FFP preparation on the MP profile. WB units were held at room temperature (RT) or combination of RT and refrigeration for up to 24hr before FFP preparation. The MP content in thawed FFP was measured to reflect transfusion practice. The absolute number of MPs in FFP increased with longer WB hold time. Refrigeration of WB may also promote increased generation of MPs. In particular the number of platelet-derived and phosphatidylserine-containing MPs, which are known to have procoagulant properties, increased. Lipid peroxidation increased with longer WB-hold time. Donor-related factors appear to govern lipid peroxidation levels. Holistic proteomic and coagulant analyses of FFP MPs is warranted. Such information could guide the choice of the optimal handling conditions of WB and the most relevant quality control procedures for FFP. PMID:22813882

  18. iTRAQ-based proteomic analysis of plasma reveals abnormalities in lipid metabolism proteins in chronic kidney disease-related atherosclerosis

    PubMed Central

    Luczak, Magdalena; Formanowicz, Dorota; Marczak, Łukasz; Suszyńska-Zajczyk, Joanna; Pawliczak, Elżbieta; Wanic-Kossowska, Maria; Stobiecki, Maciej

    2016-01-01

    Patients with chronic kidney disease (CKD) have a considerably higher risk of death due to cardiovascular causes. Using an iTRAQ MS/MS approach, we investigated the alterations in plasma protein accumulation in patients with CKD and classical cardiovascular disease (CVD) without CKD. The proteomic analysis led to the identification of 130 differentially expressed proteins among CVD and CKD patients and healthy volunteers. Bioinformatics analysis revealed that 29 differentially expressed proteins were involved in lipid metabolism and atherosclerosis, 20 of which were apolipoproteins and constituents of high-density lipoprotein (HDL) and low-density lipoprotein (LDL). Although dyslipidemia is common in CKD patients, we found that significant changes in apolipoproteins were not strictly associated with changes in plasma lipid levels. A lack of correlation between apoB and LDL concentration and an inverse relationship of some proteins with the HDL level were revealed. An increased level of apolipoprotein AIV, adiponectin, or apolipoprotein C, despite their anti-atherogenic properties, was not associated with a decrease in cardiovascular event risk in CKD patients. The presence of the distinctive pattern of apolipoproteins demonstrated in this study may suggest that lipid abnormalities in CKD are characterized by more qualitative abnormalities and may be related to HDL function rather than HDL deficiency. PMID:27600335

  19. Mouse-Specific Tandem IgY7-SuperMix Immunoaffinity Separations for Improved LC-MS/MS Coverage of the Plasma Proteome

    PubMed Central

    Zhou, Jian-Ying; Petritis, Brianne O.; Petritis, Konstantinos; Norbeck, Angela D.; Weitz, Karl K.; Moore, Ronald J.; Camp, David G.; Kulkarni, Rohit N.; Smith, Richard D.; Qian, Wei-Jun

    2009-01-01

    We report on a mouse specific SuperMix immunoaffinity separation system for separating low abundance proteins from high and moderate abundance proteins in mouse plasma. When applied in tandem with a mouse IgY7 column that removes the seven most abundant proteins in plasma, the SuperMix column captures more than 100 additional moderate abundance proteins, thus allowing significant enrichment of low abundance proteins in the flow-through fraction. A side-by-side comparison of results obtained from 2D-LC-MS/MS analyses of flow-through samples from IgY7 and SuperMix columns revealed a nearly two-fold improvement in the overall proteome coverage. Detection of low abundance proteins was also enhanced, as evidenced by a more than two-fold increase in the coverage of cytokines, growth factors, and other low abundance proteins. Moreover, the tandem separations are automated, reproducible, and allow effective identification of protein abundance differences from LC-MS/MS analyses. Considering the overall reproducibility and increased sensitivity using the IgY7-SuperMix separation system, we anticipate broad applications of this strategy for biomarker discovery using mouse models. PMID:19722698

  20. Proteomic Analysis of Plasma-Purified VLDL, LDL, and HDL Fractions from Atherosclerotic Patients Undergoing Carotid Endarterectomy: Identification of Serum Amyloid A as a Potential Marker

    PubMed Central

    Lepedda, Antonio J.; Zinellu, Elisabetta; De Muro, Pierina; Guarino, Anna; Spirito, Rita; Carta, Franco; Turrini, Francesco

    2013-01-01

    Apolipoproteins are very heterogeneous protein family, implicated in plasma lipoprotein structural stabilization, lipid metabolism, inflammation, or immunity. Obtaining detailed information on apolipoprotein composition and structure may contribute to elucidating lipoprotein roles in atherogenesis and to developing new therapeutic strategies for the treatment of lipoprotein-associated disorders. This study aimed at developing a comprehensive method for characterizing the apolipoprotein component of plasma VLDL, LDL, and HDL fractions from patients undergoing carotid endarterectomy, by means of two-dimensional electrophoresis (2-DE) coupled with Mass Spectrometry analysis, useful for identifying potential markers of plaque presence and vulnerability. The adopted method allowed obtaining reproducible 2-DE maps of exchangeable apolipoproteins from VLDL, LDL, and HDL. Twenty-three protein isoforms were identified by peptide mass fingerprinting analysis. Differential proteomic analysis allowed for identifying increased levels of acute-phase serum amyloid A protein (AP SAA) in all lipoprotein fractions, especially in LDL from atherosclerotic patients. Results have been confirmed by western blotting analysis on each lipoprotein fraction using apo AI levels for data normalization. The higher levels of AP SAA found in patients suggest a role of LDL as AP SAA carrier into the subendothelial space of artery wall, where AP SAA accumulates and may exert noxious effects. PMID:24454983

  1. Proteomic analysis of plasma-purified VLDL, LDL, and HDL fractions from atherosclerotic patients undergoing carotid endarterectomy: identification of serum amyloid A as a potential marker.

    PubMed

    Lepedda, Antonio J; Nieddu, Gabriele; Zinellu, Elisabetta; De Muro, Pierina; Piredda, Franco; Guarino, Anna; Spirito, Rita; Carta, Franco; Turrini, Francesco; Formato, Marilena

    2013-01-01

    Apolipoproteins are very heterogeneous protein family, implicated in plasma lipoprotein structural stabilization, lipid metabolism, inflammation, or immunity. Obtaining detailed information on apolipoprotein composition and structure may contribute to elucidating lipoprotein roles in atherogenesis and to developing new therapeutic strategies for the treatment of lipoprotein-associated disorders. This study aimed at developing a comprehensive method for characterizing the apolipoprotein component of plasma VLDL, LDL, and HDL fractions from patients undergoing carotid endarterectomy, by means of two-dimensional electrophoresis (2-DE) coupled with Mass Spectrometry analysis, useful for identifying potential markers of plaque presence and vulnerability. The adopted method allowed obtaining reproducible 2-DE maps of exchangeable apolipoproteins from VLDL, LDL, and HDL. Twenty-three protein isoforms were identified by peptide mass fingerprinting analysis. Differential proteomic analysis allowed for identifying increased levels of acute-phase serum amyloid A protein (AP SAA) in all lipoprotein fractions, especially in LDL from atherosclerotic patients. Results have been confirmed by western blotting analysis on each lipoprotein fraction using apo AI levels for data normalization. The higher levels of AP SAA found in patients suggest a role of LDL as AP SAA carrier into the subendothelial space of artery wall, where AP SAA accumulates and may exert noxious effects.

  2. Proteomic analysis in cardiovascular research.

    PubMed

    Oda, Teiji; Matsumoto, Ken-ichi

    2016-03-01

    Advances in mass spectrometry technology and bioinformatics using clinical human samples have expanded quantitative proteomics in cardiovascular research. There are two major proteomic strategies: namely, "gel-based" or "gel-free" proteomics coupled with either "top-down" or "bottom-up" mass spectrometry. Both are introduced into the proteomic analysis using plasma or serum sample targeting 'biomarker" searches of aortic aneurysm and tissue samples, such as from the aneurysmal wall, calcific aortic valve, or myocardial tissue, investigating pathophysiological protein interactions and post-translational modifications. We summarize the proteomic studies that analyzed human samples taken during cardiovascular surgery to investigate disease processes, in order to better understand the system-wide changes behind known molecular factors and specific signaling pathways.

  3. Reference distribution functions for magnetically confined plasmas from the minimum entropy production theorem and the MaxEnt principle, subject to the scale-invariant restrictions

    NASA Astrophysics Data System (ADS)

    Sonnino, Giorgio; Cardinali, Alessandro; Steinbrecher, Gyorgy; Peeters, Philippe; Sonnino, Alberto; Nardone, Pasquale

    2013-12-01

    We derive the expression of the reference distribution function for magnetically confined plasmas far from the thermodynamic equilibrium. The local equilibrium state is fixed by imposing the minimum entropy production theorem and the maximum entropy (MaxEnt) principle, subject to scale invariance restrictions. After a short time, the plasma reaches a state close to the local equilibrium. This state is referred to as the reference state. The aim of this Letter is to determine the reference distribution function (RDF) when the local equilibrium state is defined by the above mentioned principles. We prove that the RDF is the stationary solution of a generic family of stochastic processes corresponding to an universal Landau-type equation with white parametric noise. As an example of application, we consider a simple, fully ionized, magnetically confined plasmas, with auxiliary Ohmic heating. The free parameters are linked to the transport coefficients of the magnetically confined plasmas, by the kinetic theory.

  4. Definitive Characterization of CA 19-9 in Resectable Pancreatic Cancer Using a Reference Set of Serum and Plasma Specimens.

    PubMed

    Haab, Brian B; Huang, Ying; Balasenthil, Seetharaman; Partyka, Katie; Tang, Huiyuan; Anderson, Michelle; Allen, Peter; Sasson, Aaron; Zeh, Herbert; Kaul, Karen; Kletter, Doron; Ge, Shaokui; Bern, Marshall; Kwon, Richard; Blasutig, Ivan; Srivastava, Sudhir; Frazier, Marsha L; Sen, Subrata; Hollingsworth, Michael A; Rinaudo, Jo Ann; Killary, Ann M; Brand, Randall E

    2015-01-01

    The validation of candidate biomarkers often is hampered by the lack of a reliable means of assessing and comparing performance. We present here a reference set of serum and plasma samples to facilitate the validation of biomarkers for resectable pancreatic cancer. The reference set includes a large cohort of stage I-II pancreatic cancer patients, recruited from 5 different institutions, and relevant control groups. We characterized the performance of the current best serological biomarker for pancreatic cancer, CA 19-9, using plasma samples from the reference set to provide a benchmark for future biomarker studies and to further our knowledge of CA 19-9 in early-stage pancreatic cancer and the control groups. CA 19-9 distinguished pancreatic cancers from the healthy and chronic pancreatitis groups with an average sensitivity and specificity of 70-74%, similar to previous studies using all stages of pancreatic cancer. Chronic pancreatitis patients did not show CA 19-9 elevations, but patients with benign biliary obstruction had elevations nearly as high as the cancer patients. We gained additional information about the biomarker by comparing two distinct assays. The two CA 9-9 assays agreed well in overall performance but diverged in measurements of individual samples, potentially due to subtle differences in antibody specificity as revealed by glycan array analysis. Thus, the reference set promises be a valuable resource for biomarker validation and comparison, and the CA 19-9 data presented here will be useful for benchmarking and for exploring relationships to CA 19-9.

  5. Definitive Characterization of CA 19-9 in Resectable Pancreatic Cancer Using a Reference Set of Serum and Plasma Specimens

    PubMed Central

    Haab, Brian B.; Huang, Ying; Balasenthil, Seetharaman; Partyka, Katie; Tang, Huiyuan; Anderson, Michelle; Allen, Peter; Sasson, Aaron; Zeh, Herbert; Kaul, Karen; Kletter, Doron; Ge, Shaokui; Bern, Marshall; Kwon, Richard; Blasutig, Ivan; Srivastava, Sudhir; Frazier, Marsha L.; Sen, Subrata; Hollingsworth, Michael A.; Rinaudo, Jo Ann; Killary, Ann M.; Brand, Randall E.

    2015-01-01

    The validation of candidate biomarkers often is hampered by the lack of a reliable means of assessing and comparing performance. We present here a reference set of serum and plasma samples to facilitate the validation of biomarkers for resectable pancreatic cancer. The reference set includes a large cohort of stage I-II pancreatic cancer patients, recruited from 5 different institutions, and relevant control groups. We characterized the performance of the current best serological biomarker for pancreatic cancer, CA 19–9, using plasma samples from the reference set to provide a benchmark for future biomarker studies and to further our knowledge of CA 19–9 in early-stage pancreatic cancer and the control groups. CA 19–9 distinguished pancreatic cancers from the healthy and chronic pancreatitis groups with an average sensitivity and specificity of 70–74%, similar to previous studies using all stages of pancreatic cancer. Chronic pancreatitis patients did not show CA 19–9 elevations, but patients with benign biliary obstruction had elevations nearly as high as the cancer patients. We gained additional information about the biomarker by comparing two distinct assays. The two CA 9–9 assays agreed well in overall performance but diverged in measurements of individual samples, potentially due to subtle differences in antibody specificity as revealed by glycan array analysis. Thus, the reference set promises be a valuable resource for biomarker validation and comparison, and the CA 19–9 data presented here will be useful for benchmarking and for exploring relationships to CA 19–9. PMID:26431551

  6. EXPLORATORY PLASMA BIOCHEMISTRY REFERENCE INTERVALS FOR URAL OWLS (STRIX URALENSIS, PALLAS 1771) FROM THE AUSTRIAN REINTRODUCTION PROJECT.

    PubMed

    Scope, Alexandra; Schwendenwein, Ilse; Stanclova, Gabriela; Vobornik, Angela; Zink, Richard

    2016-06-01

    The Ural owl (Strix uralensis) is the biggest forest-living owl in Austria; however, it became extinct in Austria through poaching and habitat loss more than half a century ago. The birds examined in the present study were breeding pairs from the reintroduction project with the aim of determining exploratory plasma biochemistry reference intervals in Ural owls and evaluating the amount of biological variation between seasons, sexes, and ages. A total of 45 birds were sampled, including 13 adult males, 14 adult females, and 18 juvenile birds. Remarkably, almost all of the analytes showed significant differences between the subgroups, primarily between seasons, followed by age and sex. Only creatinkinase, glucose, lactatdehydrogenase, and triglycerides did not show any significant variations. Despite partitioning of reference values into subgroups according to biological variation diminishing the number of reference individuals in the respective groups, the resulting smaller reference intervals will improve medical assessment. The results of the present study once again demonstrate that significant seasonal fluctuations must be expected and considered in the interpretation. It can be assumed that these differences are probably even greater in free-range birds with considerable changes in food quantity and quality during and between years.

  7. Plasma membrane proteomics of tumor spheres identify CD166 as a novel marker for cancer stem-like cells in head and neck squamous cell carcinoma.

    PubMed

    Yan, Ming; Yang, Xihu; Wang, Lizhen; Clark, David; Zuo, Hui; Ye, Dongxia; Chen, Wantao; Zhang, Ping

    2013-11-01

    Patients with advanced head and neck squamous cell carcinoma (HNSCC) have a poor prognosis with the currently available therapy, and tumor recurrence is frequently observed. The discovery of specific membrane-associated cancer stem cell (CSC) markers is crucial for the development of novel therapeutic strategies to target these CSCs. To address this issue, we established sphere cultures to enrich CSCs and used them for plasma membrane proteomics to identify specific membrane signatures of the HNSCC spheres. Of a dataset that included a total of 376 identified proteins, 200 were bona fide membrane proteins. Among them, 123 proteins were at least 1.5-fold up- or down-regulated in the spheres relative to the adherent cultures. These proteins included cell adhesion molecules, receptors, and transporter proteins. Some of them play key roles in wnt, integrin, and TGFβ signaling pathways. When we compared our dataset with two published hESC membrane protein signatures, we found 18 proteins common to all three of the databases. CD166 and CD44 were two such proteins. Interestingly, the expression of CD166, rather than that of the well-established HNSCC CSC marker CD44, was significantly related to the malignant behavior of HNSCC. Relative to CD166(low) HNSCC cells, CD166(high) HNSCC cells had a greater sphere-formation ability in vitro and tumor formation ability in vivo. Patients whose tumors expressed high levels of CD166 had a significantly poorer clinical outcome than those whose tumors expressed low levels of CD166 (cohort 1: 96 cases, p = 0.040), whereas the level of CD44 expression had only a marginal influence on the clinical outcome of patients with HNSCC (p = 0.078). The level of CD166 expression in HNSCC tumors was also associated with the tumor recurrence rate (cohort 2: 104 cases, p = 0.016). This study demonstrates that CD166 is a valuable cell surface marker for the enrichment of HNSCC stem cells and that plasma membrane proteomics is a promising biological

  8. Proteomics of plasma membranes from poplar trees reveals tissue distribution of transporters, receptors, and proteins in cell wall formation.

    PubMed

    Nilsson, Robert; Bernfur, Katja; Gustavsson, Niklas; Bygdell, Joakim; Wingsle, Gunnar; Larsson, Christer

    2010-02-01

    By exploiting the abundant tissues available from Populus trees, 3-4 m high, we have been able to isolate plasma membranes of high purity from leaves, xylem, and cambium/phloem at a time (4 weeks after bud break) when photosynthesis in the leaves and wood formation in the xylem should have reached a steady state. More than 40% of the 956 proteins identified were found in the plasma membranes of all three tissues and may be classified as "housekeeping" proteins, a typical example being P-type H(+)-ATPases. Among the 213 proteins predicted to be integral membrane proteins, transporters constitute the largest class (41%) followed by receptors (14%) and proteins involved in cell wall and carbohydrate metabolism (8%) and membrane trafficking (8%). ATP-binding cassette transporters (all members of subfamilies B, C, and G) and receptor-like kinases (four subfamilies) were two of the largest protein families found, and the members of these two families showed pronounced tissue distribution. Leaf plasma membranes were characterized by a very high proportion of transporters, constituting almost half of the integral proteins. Proteins involved in cell wall synthesis (such as cellulose and sucrose synthases) and membrane trafficking were most abundant in xylem plasma membranes in agreement with the role of the xylem in wood formation. Twenty-five integral proteins and 83 soluble proteins were exclusively found in xylem plasma membranes, which identifies new candidates associated with cell wall synthesis and wood formation. Among the proteins uniquely found in xylem plasma membranes were most of the enzymes involved in lignin biosynthesis, which suggests that they may exist as a complex linked to the plasma membrane.

  9. Reference intervals of plasma calcium, phosphorus, and magnesium for African grey parrots (Psittacus erithacus) and Hispaniolan parrots (Amazona ventralis).

    PubMed

    de Carvalho, Fernanda M; Gaunt, Stephen D; Kearney, Michael T; Rich, Gregory A; Tully, Thomas N

    2009-12-01

    Calcium (Ca), phosphorus (P), and magnesium (Mg) are important elements for body homeostasis in several diseases associated with imbalances in the plasma concentration of these ions. This is the first published report of reference intervals for Mg in association with Ca and P levels for psittacine species. One milliliter of blood was collected from 26 Hispaniolan parrots (Amazona ventralis) and 24 African grey parrots (Psittacus erithacus). The plasma concentrations of Ca, P, and Mg were determined for each sample. Statistical analyses were performed including all data (analysis 1) and after exclusion of the subjects with Ca > or = 14.00 mg/dl (3.5 mmol) (analysis 2). The data from analysis 1 have a narrower interval than that observed in analysis 2. Following the normality test (Shapiro-Wilk, alpha = 0.05), the univariate and mean procedures were run. For the reference intervals, the lower and upper values were used, after elimination of the outliers calculated by Blom scores from the ranked variables. The analysis 1 references for the Hispaniolans were Ca = 8.80-10.40 mg/dl (2.20-2.60 mmol/L), P = 1.80-4.40 mg/dl (0.58-1.42 mmol/L), Mg = 1.80-3.10 mg/dl (0.74-1.27 mmol/L), and Ca:P ratio = 2.62-5.39; for the African greys analysis 1 references were Ca = 8.20-20.20 mg/dl (2.05-5.05 mmol/L), P = 2.50-5.90 mg/dl (0.81-1.91 mmol/L), Mg = 2.10-3.40 mg/dl (0.82-1.4 mmol/L), and Ca:P ratio = 1.81-3.77. The analysis 2 references for the Hispaniolans were Ca = 8.80-10.30 mg/dl (2.20-2.58 mmol/L), P = 1.80-3.80 mg/dl (0.58-1.23 mmol/L), Mg = 1.90-3.00 mg/dl (0.82-1.07 mmol/L), Ca:P ratio = 2.62-5.39; for the African greys analysis 2 references were Ca = 1.07 mmol/L), Ca:P ratio = 1.67-3.50. The results of this study are important for evaluating Mg concentrations in relation to the Ca and P parameters in psittacines. This information will be particularly helpful for veterinarians evaluating the hypocalcemic syndrome in African grey parrots and other disease processes

  10. BRSCW Reference Set Application: Karen Abbott -University of Arkansas (2014) — EDRN Public Portal

    Cancer.gov

    Our earlier glycoproteomic studies have identified bisecting glycoslyation and core fucosylation changes on particular glycoproteins in endometrioid ovarian cancer tissues and plasma (Abbott et al, 2010, Proteomics). We have validated that these glycan changes occur on the same glycoproteins in serous ovarian cancer plasma using a lectin-pull down western blot assays. We would like to used pooled reference samples to develop a sensitive magnetic bead-based assay to detect these glycoproteins with bisecting and core fucosylation changes.

  11. Proteomic analysis of seminal plasma from asthenozoospermia patients reveals proteins that affect oxidative stress responses and semen quality.

    PubMed

    Wang, Jun; Wang, Jian; Zhang, Hua-Rong; Shi, Hui-Juan; Ma, Duan; Zhao, Hong-Xin; Lin, Biaoyang; Li, Run-Sheng

    2009-07-01

    Asthenozoospermia (AS) is a common cause of human male infertility. In one study, more than 80% of the samples from infertile men had reduced sperm motility. Seminal plasma is a mixture of secretions from the testis, epididymis and several male accessory glands, including the prostate, seminal vesicles and Cowper's gland. Studies have shown that seminal plasma contains proteins that are important for sperm motility. To further explore the pathophysiological character of AS, we separated the seminal plasma proteins from AS patients and healthy donors using sodium dodecyl sulfate polyacrylamide gel electrophoresis and in-gel digestion, and then subjected the proteins to liquid chromatography-mass spectrometry (LC-MS/MS) analysis. A total of 741 proteins were identified in the seminal plasma, with a false discovery rate of 3.3%. Using spectral counting, we found that 45 proteins were threefold upregulated and 56 proteins were threefold downregulated in the AS group when compared with the control. Most of these proteins originated from the epididymis and prostate. This study identified a rich source of biomarker candidates for male infertility and indicates that functional abnormalities of the epididymis and prostate can contribute to AS. We identified DJ-1-a protein that has been shown elsewhere to be involved in the control of oxidative stress (OS)-as a downregulated protein in AS seminal plasma. The levels of DJ-1 in AS seminal plasma were about half of those in the control samples. In addition, the levels of reactive oxygen species were 3.3-fold higher in the AS samples than in the controls. Taken together, these data suggest that downregulation of DJ-1 is involved in OS in semen, and therefore affects the quality of the semen.

  12. Rejuvenating rice proteomics: facts, challenges, and visions.

    PubMed

    Agrawal, Ganesh Kumar; Jwa, Nam-Soo; Iwahashi, Yumiko; Yonekura, Masami; Iwahashi, Hitoshi; Rakwal, Randeep

    2006-10-01

    Proteomics is progressing at an unprecedented pace, as can be exemplified by the progress in model organisms such as yeast, bacteria, and mammals. Proteomics research in plants, however, has not progressed at the same pace. Unscrambling of the genome sequences of the dicotyledoneous Arabidopsis thaliana (L.) and monocotyledoneous rice (Oryza sativa L.) plant species, respectively, has made them accessible reference organisms to study plant proteomics. Study of these two reference plants is expected to unravel the mystery of plant biology. Rice, a critically important food crop on the earth, has been termed a "cornerstone" and the "Rosetta stone" for functional genomics of cereal crops. Here, we look at the progress in unraveling rice proteomes and present the facts, challenges, and vision. The text is divided into two major parts: the first part presents the facts and the second part discusses the challenges and vision. The facts include the technology and its use in developing proteomes, which have been critically and constructively reviewed. The challenges and vision deal with the establishment of technologies to exhaustively investigate the protein components of a proteome, to generate high-resolution gel-based reference maps, and to give rice proteomics a functional dimension by studying PTMs and isolation of multiprotein complexes. Finally, we direct a vision on rice proteomics. This is our third review in series on rice proteomics, which aims to stimulate an objective discussion among rice researchers and to understand the necessity and impact of unraveling rice proteomes to their full potential.

  13. Changes in the Plasma Proteome of Manduca sexta Larvae in Relation to the Transcriptome Variations after an Immune Challenge: Evidence for High Molecular Weight Immune Complex Formation.

    PubMed

    He, Yan; Cao, Xiaolong; Zhang, Shuguang; Rogers, Janet; Hartson, Steve; Jiang, Haobo

    2016-04-01

    Manduca sextais a lepidopteran model widely used to study insect physiological processes, including innate immunity. In this study, we explored the proteomes of cell-free hemolymph from larvae injected with a sterile buffer (C for control) or a mixture of bacteria (I for induced). Of the 654 proteins identified, 70 showed 1.67 to >200-fold abundance increases after the immune challenge; 51 decreased to 0-60% of the control levels. While there was no strong parallel between plasma protein levels and their transcript levels in hemocytes or fat body, the mRNA level changes (i.e.I/C ratios of normalized read numbers) in the tissues concurred with their protein level changes (i.e.I/C ratios of normalized spectral counts) with correlation coefficients of 0.44 and 0.57, respectively. Better correlations support that fat body contributes a more significant portion of the plasma proteins involved in various aspects of innate immunity. Consistently, ratios of mRNA and protein levels were better correlated for immunity-related proteins than unrelated ones. There is a set of proteins whose apparent molecular masses differ considerably from the calculatedMr's, suggestive of posttranslational modifications. In addition, some lowMrproteins were detected in the range of 80 to >300 kDa on a reducing SDS-polyacrylamide gel, indicating the existence of highMrcovalent complexes. We identified 30 serine proteases and their homologs, 11 of which are known members of an extracellular immune signaling network. Along with our quantitative transcriptome data, the protein identification, inducibility, and association provide leads toward a focused exploration of humoral immunity inM. sexta.

  14. Changes in the Plasma Proteome of Manduca sexta Larvae in Relation to the Transcriptome Variations after an Immune Challenge: Evidence for High Molecular Weight Immune Complex Formation*

    PubMed Central

    He, Yan; Cao, Xiaolong; Zhang, Shuguang; Rogers, Janet; Hartson, Steve; Jiang, Haobo

    2016-01-01

    Manduca sexta is a lepidopteran model widely used to study insect physiological processes, including innate immunity. In this study, we explored the proteomes of cell-free hemolymph from larvae injected with a sterile buffer (C for control) or a mixture of bacteria (I for induced). Of the 654 proteins identified, 70 showed 1.67 to >200-fold abundance increases after the immune challenge; 51 decreased to 0–60% of the control levels. While there was no strong parallel between plasma protein levels and their transcript levels in hemocytes or fat body, the mRNA level changes (i.e. I/C ratios of normalized read numbers) in the tissues concurred with their protein level changes (i.e. I/C ratios of normalized spectral counts) with correlation coefficients of 0.44 and 0.57, respectively. Better correlations support that fat body contributes a more significant portion of the plasma proteins involved in various aspects of innate immunity. Consistently, ratios of mRNA and protein levels were better correlated for immunity-related proteins than unrelated ones. There is a set of proteins whose apparent molecular masses differ considerably from the calculated Mr's, suggestive of posttranslational modifications. In addition, some low Mr proteins were detected in the range of 80 to >300 kDa on a reducing SDS-polyacrylamide gel, indicating the existence of high Mr covalent complexes. We identified 30 serine proteases and their homologs, 11 of which are known members of an extracellular immune signaling network. Along with our quantitative transcriptome data, the protein identification, inducibility, and association provide leads toward a focused exploration of humoral immunity in M. sexta. PMID:26811355

  15. Buffalo cervico-vaginal fluid proteomics with special reference to estrous cycle: heat shock protein (HSP)-70 appears to be an estrus indicator.

    PubMed

    Muthukumar, Subramanian; Rajkumar, Ramalingam; Karthikeyan, Kandasamy; Liao, Chen-Chung; Singh, Dheer; Akbarsha, Mohammad Abdulkader; Archunan, Govindaraju

    2014-05-01

    Cervico-vaginal fluid (CVF) plays significant roles in coitus, sperm transport, and implantation. It is believed to be a good noninvasive biomarker for various diagnostic purposes. In this study, a comprehensive proteomic analysis of buffalo CVF was performed during the estrous cycle in order to document the protein expressions, utilizing SDS-PAGE, mass spectrometry, and immunoblot. The main objective was to screen the CVF of buffalo for one or more estrus-specific proteins. A total of 416 proteins were identified in the CVF of both estrus and diestrus phases. Out of these proteins, 68 estrus-specific proteins have been extensively reviewed in the protein database. The major physiological functions of estrus CVF proteins appeared to be stress response, immune response, and metabolic. Eventually, the expression level of heat shock protein-70 in the CVF during the estrus phase, as revealed in SDS-PAGE analysis, was higher than during diestrus. The identity of the protein was confirmed by immunoblot analysis as heat shock protein-70. The findings provide a potential lead for the evaluation of these proteins for estrus detection in buffalo because CVF biomarker detection is a noninvasive technique. The mass spectrometric data of identified proteins have been deposited at the ProteomeXchange with the identifier PXD000620.

  16. Gene-centric view on the human proteome project: the example of the Russian roadmap for chromosome 18.

    PubMed

    Archakov, Alexander; Aseev, Alexander; Bykov, Victor; Grigoriev, Anatoly; Govorun, Vadim; Ivanov, Vadim; Khlunov, Alexander; Lisitsa, Andrey; Mazurenko, Sergey; Makarov, Alexander A; Ponomarenko, Elena; Sagdeev, Renad; Skryabin, Konstantin

    2011-05-01

    During the 2010 Human Proteome Organization Congress in Sydney, a gene-centric approach emerged as a feasible and tractable scaffold for assemblage of the Human Proteome Project. Bringing the gene-centric principle into practice, a roadmap for the 18th chromosome was drafted, postulating the limited sensitivity of analytical methods, as a serious bottleneck in proteomics. In the context of the sensitivity problem, we refer to the "copy number of protein molecules" as a measurable assessment of protein abundance. The roadmap is focused on the development of technology to attain the low- and ultralow -"copied" portion of the proteome. Roadmap merges the genomic, transcriptomic and proteomic levels to identify the majority of 285 proteins from 18th chromosome - master proteins. Master protein is the primary translation of the coding sequence and resembling at least one of the known isoforms, coded by the gene. The executive phase of the roadmap includes the expansion of the study of the master proteins with alternate splicing, single amino acid polymorphisms (SAPs) and post-translational modifications. In implementing the roadmap, Russian scientists are expecting to establish proteomic technologies for integrating MS and atomic force microscopy (AFM). These technologies are anticipated to unlock the value of new biomarkers at a detection limit of 10(-18) M, i.e. 1 protein copy per 1 μL of plasma. The roadmap plan is posted at www.proteome.ru/en/roadmap/ and a forum for discussion of the document is supported.

  17. Modifications of plasma proteome in long-lived rats fed on a coenzyme Q10-supplemented diet.

    PubMed

    Santos-González, Mónica; Gómez Díaz, Consuelo; Navas, Plácido; Villalba, José Manuel

    2007-08-01

    Dietary coenzyme Q(10) prolongs life span of rats fed on a PUFAn-6-enriched diet. Our aim was to analyze changes in the levels of plasma proteins of rats fed on a PUFAn-6 plus coenzyme Q(10)-based diet. This approach could give novel insights into the mechanisms of life span extension by dietary coenzyme Q(10) in the rat. Serum albumin, which decreases with aging in the rat, was significantly increased by coenzyme Q(10) supplementation both at 6 and 24 months. After depletion of the most abundant proteins by affinity chromatography, levels of less abundant plasma proteins were also studied by using 2D-electrophoresis and MALDI-TOF mass fingerprinting analysis. Our results have shown that lifelong dietary supplementation with coenzyme Q(10) induced significant decreases of plasma hemopexin, apolipoprotein H and inter-alpha-inhibitor H4P heavy chain (at both 6 and 24 months), preprohaptoglobin, fibrinogen gamma-chain precursor, and fetuin-like protein (at 6 months), and alpha-1-antitrypsin precursor and type II peroxiredoxin (at 24 months). On the other hand, coenzyme Q(10) supplementation resulted in significant increases of serine protease inhibitor 3, vitamin D-binding protein (at 6 months), and Apo A-I (at 24 months). Our results support a beneficial role of dietary coenzyme Q(10) decreasing oxidative stress and cardiovascular risk, and modulating inflammation during aging.

  18. Determination of organomercury in biological reference materials by inductively coupled plasma mass spectrometry using flow injection analysis

    SciTech Connect

    Beauchemin, D.; Siu, K.W.; Berman, S.S.

    1988-12-01

    Inductively coupled plasma mass spectrometry was used for the determination of organomercury in two marine biological standard reference materials for trace metals (dogfish muscle tissue DORM-1 and lobster hepatopancreas TORT-1). In most parts of this study, the organomercury was extracted as the chloride from the material with toluene and back extracted into an aqueous medium of cysteine acetate. Since the final extracts contained more than 4% sodium, isotope dilution and flow injection analysis were used to respectively counter the effect of concomitant elements and avoid clogging the interface. Comparison of results with gas chromatography shows that the only significant organomercury is methyl-mercury. At least 93% of mercury in DORM-1 and 39% of mercury in TORT-1 exist as methylmercury.

  19. Screening for phaeochromocytoma and paraganglioma: impact of using supine reference intervals for plasma metanephrines with samples collected from fasted/seated patients.

    PubMed

    Casey, R; Griffin, T P; Wall, D; Dennedy, M C; Bell, M; O'Shea, P M

    2017-01-01

    Background The Endocrine Society Clinical Practice Guideline on Phaeochomocytoma and Paraganglioma recommends phlebotomy for plasma-free metanephrines with patients fasted and supine using appropriately defined reference intervals. Studies have shown higher diagnostic sensitivities using these criteria. Further, with seated-sampling protocols, for result interpretation, reference intervals that do not compromise diagnostic sensitivity should be employed. Objective To determine the impact on diagnostic performance and financial cost of using supine reference intervals for result interpretation with our current plasma-free metanephrines fasted/seated-sampling protocol. Methods We conducted a retrospective cohort study of patients who underwent screening for PPGL using plasma-free metanephrines from 2009 to 2014 at Galway University Hospitals. Plasma-free metanephrines were measured using liquid chromatography-tandem mass spectrometry. Supine thresholds for plasma normetanephrine and metanephrine set at 610 pmol/L and 310 pmol/L, respectively, were used. Results A total of 183 patients were evaluated. Mean age of participants was 53.4 (±16.3) years. Five of 183 (2.7%) patients had histologically confirmed PPGL (males, n=4). Using seated reference intervals for plasma-free metanephrines, diagnostic sensitivity and specificity were 100% and 98.9%, respectively, with two false-positive cases. Application of reference intervals established in subjects supine and fasted to this cohort gave diagnostic sensitivity of 100% with specificity of 74.7%. Financial analysis of each pretesting strategy demonstrated cost-equivalence (€147.27/patient). Conclusion Our cost analysis, together with the evidence that fasted/supine-sampling for plasma-free metanephrines, offers more reliable exclusion of PPGL mandates changing our current practice. This study highlights the important advantages of standardized diagnostic protocols for plasma-free metanephrines to ensure the highest

  20. In-depth analysis of the human tear proteome.

    PubMed

    Zhou, Lei; Zhao, Shao Zhen; Koh, Siew Kwan; Chen, Liyan; Vaz, Candida; Tanavde, Vivek; Li, Xiao Rong; Beuerman, Roger W

    2012-07-16

    The tears, a critical body fluid of the surface of the eye, contain an unknown number of molecules including proteins/peptides, lipids, small molecule metabolites, and electrolytes. There have been continued efforts for exploring the human tear proteome to develop biomarkers of disease. In this study, we used the high speed TripleTOF 5600 system as the platform to analyze the human tear proteome from healthy subjects (3 females and 1 male, average age: 36±14). We have identified 1543 proteins in the tears with less than 1% false discovery rate, which represents the largest number of human tear proteins reported to date. The data set was analyzed for gene ontology (GO) and compared with the human plasma proteome, NEIBank lacrimal gland gene dataset and NEIBank cornea gene dataset. This comprehensive tear protein list may serve as a reference list of human tear proteome for biomarker research of ocular diseases or establishment of MRM (Multiple Reaction Monitoring) assays for targeted analysis. Tear fluid is a useful and an accessible source not only for evaluating ocular surface tissues (cornea and conjunctiva), inflammation, lacrimal gland function and a number of disease conditions, such as dry eye as well as response to treatment. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. Harmonizing Lipidomics: NIST Interlaboratory Comparison Exercise for Lipidomics using Standard Reference Material 1950 Metabolites in Frozen Human Plasma.

    PubMed

    Bowden, John A; Heckert, Alan; Ulmer, Candice Z; Jones, Christina M; Koelmel, Jeremy P; Abdullah, Laila; Ahonen, Linda; Alnouti, Yazen; Armando, Aaron; Asara, John M; Bamba, Takeshi; Barr, John R; Bergquist, Jonas; Borchers, Christoph H; Brandsma, Joost; Breitkopf, Susanne B; Cajka, Tomas; Cazenave-Gassiot, Amaury; Checa, Antonio; Cinel, Michelle A; Colas, Romain A; Cremers, Serge; Dennis, Edward A; Evans, James E; Fauland, Alexander; Fiehn, Oliver; Gardner, Michael S; Garrett, Timothy J; Gotlinger, Katherine H; Han, Jun; Huang, Yingying; Neo, Aveline Huipeng; Hyotylainen, Tuulia; Izumi, Yoshihiro; Jiang, Hongfeng; Jiang, Houli; Jiang, Jiang; Kachman, Maureen; Kiyonami, Reiko; Klavins, Kristaps; Klose, Christian; Kofeler, Harald C; Kolmert, Johan; Koal, Therese; Koster, Grielof; Kuklenyik, Zsuzsanna; Kurland, Irwin J; Leadley, Michael; Lin, Karen; Maddipati, Krishna Rao; McDougall, Danielle; Meikle, Peter J; Mellett, Natalie A; Monnin, Cian; Moseley, M Arthur; Nandakumar, Renu; Oresic, Matej; Patterson, Rainey E; Peake, David; Pierce, Jason S; Post, Martin; Postle, Anthony D; Pugh, Rebecca; Qui, Yunping; Quehenberger, Oswald; Ramrup, Parsram; Rees, Jon; Rembiesa, Barbara; Reynaud, Denis; Roth, Mary R; Sales, Susanne; Schuhmann, Kai; Schwartzman, Michal Laniado; Serhan, Charles N; Shevchenko, Andrej; Somerville, Stephen E; John-Williams, Lisa St; Surma, Michal A; Takeda, Hiroaki; Thakare, Rhishikesh; Thompson, J Will; Torta, Federico; Triebl, Alexander; Trotzmuller, Martin; Ubhayasekera, S J Kumari; Vuckovic, Dajana; Weir, Jacquelyn M; Welti, Ruth; Wenk, Markus R; Wheelock, Craig E; Yao, Libin; Yuan, Min; Zhao, Xueqing Heather; Zhou, Senlin

    2017-10-06

    As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950 Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each lab using a different lipidomics workflow. A total of 1527 unique lipids were measured across all laboratories, and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and inter-laboratory quality control and method validation. These analyses were performed using non-standardized laboratory-independent workflows. The consensus locations were also compared to a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  2. Oolemmal proteomics--identification of highly abundant heat shock proteins and molecular chaperones in the mature mouse egg and their localization on the plasma membrane.

    PubMed

    Calvert, Meredith E; Digilio, Laura C; Herr, John C; Coonrod, Scott A

    2003-02-14

    The mature mouse egg contains the full complement of maternal proteins required for fertilization, the transition to zygotic transcription, and the beginning stages of embryogenesis. Many of these proteins remain to be characterized, therefore in this study we have identified highly abundant egg proteins using a proteomic approach and found that several of these proteins also appear to localize to the egg surface. Characterization of such molecules will provide important insight into the cellular events of fertilization and early development. In order to identify some of the more abundant egg proteins, whole egg extracts were resolved on coomassie-stained two-dimensional (2D) PAGE gels. Several highly abundant protein spots were cored and microsequenced by tandem mass spectrometry (TMS), and determined to be molecular chaperone proteins. Concurrent experiments were performed to identify oolemmal proteins using 2D avidin blotting. Proteins spots that appeared to be surface labeled by biotinylation were correlated with the initial coomassie-stained reference gel. Surprisingly, some of the surface labelled proteins corresponded to those abundant chaperone proteins previously identified. To confirm whether these molecules are accumulating at the oolemmal surface in eggs, we performed immunofluoresence on live, zona-free eggs using antibodies to HSP70, HSP90, GRP94, GRP78, calreticulin and calnexin. The putative surface-labeled proteins identified by biotinylation included the molecular chaperones HSP70 (MW 70 KDa, pI 5.5), HSP90a (MW 85 KDa, pI 4.9), GRP94 (MW 92 KDa, pI 4.7), GRP78 (MW 72 KDa, pI 5.0), Oxygen regulated protein 150 (ORP150; MW 111 KDa, pI 5.1), Calreticulin (MW 48 KDa, pI 4.3), Calnexin (MW 65 KDa, pI 4.5), and Protein disulfide isomerase (PDI; MW 57 KDa, pI 4.8). Immunofluoresence results showed that antibodies to HSP90, GRP94, GRP78 and calreticulin were reactive with oolemmal proteins. We were unable to confirm surface localization of HSP70 or

  3. Oolemmal proteomics – identification of highly abundant heat shock proteins and molecular chaperones in the mature mouse egg and their localization on the plasma membrane

    PubMed Central

    Calvert, Meredith E; Digilio, Laura C; Herr, John C; Coonrod, Scott A

    2003-01-01

    Background The mature mouse egg contains the full complement of maternal proteins required for fertilization, the transition to zygotic transcription, and the beginning stages of embryogenesis. Many of these proteins remain to be characterized, therefore in this study we have identified highly abundant egg proteins using a proteomic approach and found that several of these proteins also appear to localize to the egg surface. Characterization of such molecules will provide important insight into the cellular events of fertilization and early development. Methods In order to identify some of the more abundant egg proteins, whole egg extracts were resolved on coomassie-stained two-dimensional (2D) PAGE gels. Several highly abundant protein spots were cored and microsequenced by tandem mass spectrometry (TMS), and determined to be molecular chaperone proteins. Concurrent experiments were performed to identify oolemmal proteins using 2D avidin blotting. Proteins spots that appeared to be surface labeled by biotinylation were correlated with the initial coomassie-stained reference gel. Surprisingly, some of the surface labelled proteins corresponded to those abundant chaperone proteins previously identified. To confirm whether these molecules are accumulating at the oolemmal surface in eggs, we performed immunofluoresence on live, zona-free eggs using antibodies to HSP70, HSP90, GRP94, GRP78, calreticulin and calnexin. Results The putative surface-labeled proteins identified by biotinylation included the molecular chaperones HSP70 (MW 70 KDa, pI 5.5), HSP90a (MW 85 KDa, pI 4.9), GRP94 (MW 92 KDa, pI 4.7), GRP78 (MW 72 KDa, pI 5.0), Oxygen regulated protein 150 (ORP150; MW 111 KDa, pI 5.1), Calreticulin (MW 48 KDa, pI 4.3), Calnexin (MW 65 KDa, pI 4.5), and Protein disulfide isomerase (PDI; MW 57 KDa, pI 4.8). Immunofluoresence results showed that antibodies to HSP90, GRP94, GRP78 and calreticulin were reactive with oolemmal proteins. We were unable to confirm surface

  4. Up-Regulation of Antioxidant Proteins in the Plasma Proteome during Saturation Diving: Unique Coincidence under Hypobaric Hypoxia

    PubMed Central

    Domoto, Hideharu; Iwaya, Keiichi; Ikomi, Fumitaka; Matsuo, Hirotaka; Tadano, Yutaka; Fujii, Shigenori; Tachi, Kazuyoshi; Itoh, Yoshiyuki; Sato, Michiya; Inoue, Kimitoshi; Shinomiya, Nariyoshi

    2016-01-01

    Saturation diving (SD) is one of the safest techniques for tolerating hyperbaric conditions for long durations. However, the changes in the human plasma protein profile that occur during SD are unknown. To identify differential protein expression during or after SD, 65 blood samples from 15 healthy Japanese men trained in SD were analyzed by two-dimensional fluorescence difference gel electrophoresis. The expression of two proteins, one 32.4 kDa with an isoelectric point (pI) of 5.8 and the other 44.8 kDa with pI 4.0, were elevated during SD to 60, 100, and 200 meters sea water (msw). The expression of these proteins returned to pre-diving level when the SD training was completed. The two proteins were identified using in-gel digestion and mass spectrometric analysis; the 32.4 kDa protein was transthyretin and the 44.8 kDa protein was alpha-1-acid glycoprotein 1. Oxidation was detected at methionine 13 of transthyretin and at methionine 129 of alpha-1-acid glycoprotein 1 by tandem mass spectrometry. Moreover, haptoglobin was up-regulated during the decompression phase of 200 msw. These plasma proteins up-regulated during SD have a common function as anti-oxidants. This suggests that by coordinating their biological effects, these proteins activate a defense mechanism to counteract the effects of hyperbaric-hyperoxic conditions during SD. PMID:27741252

  5. Changes in the plasma proteome at asymptomatic and symptomatic stages of autosomal dominant Alzheimer’s disease

    PubMed Central

    Muenchhoff, Julia; Poljak, Anne; Thalamuthu, Anbupalam; Gupta, Veer B.; Chatterjee, Pratishtha; Raftery, Mark; Masters, Colin L.; Morris, John C.; Bateman, Randall J.; Fagan, Anne M.; Martins, Ralph N.; Sachdev, Perminder S.

    2016-01-01

    The autosomal dominant form of Alzheimer’s disease (ADAD) is far less prevalent than late onset Alzheimer’s disease (LOAD), but enables well-informed prospective studies, since symptom onset is near certain and age of onset is predictable. Our aim was to discover plasma proteins associated with early AD pathology by investigating plasma protein changes at the asymptomatic and symptomatic stages of ADAD. Eighty-one proteins were compared across asymptomatic mutation carriers (aMC, n = 15), symptomatic mutation carriers (sMC, n = 8) and related noncarriers (NC, n = 12). Proteins were also tested for associations with cognitive measures, brain amyloid deposition and glucose metabolism. Fewer changes were observed at the asymptomatic than symptomatic stage with seven and 16 proteins altered significantly in aMC and sMC, respectively. This included complement components C3, C5, C6, apolipoproteins A-I, A-IV, C-I and M, histidine-rich glycoprotein, heparin cofactor II and attractin, which are involved in inflammation, lipid metabolism and vascular health. Proteins involved in lipid metabolism differed only at the symptomatic stage, whereas changes in inflammation and vascular health were evident at asymptomatic and symptomatic stages. Due to increasing evidence supporting the usefulness of ADAD as a model for LOAD, these proteins warrant further investigation into their potential association with early stages of LOAD. PMID:27381087

  6. Molecular biology tools: proteomics techniques in biomarker discovery.

    PubMed

    Lottspeich, Friedrich; Kellermann, Josef; Keidel, Eva-Maria

    2010-01-01

    Despite worldwide efforts biomarker discovery by plasma proteomics was not successful so far. Several reasons for this failure are obvious. Mainly, proteome diversity is remarkable between different individuals and is caused by genetic, environmental and life style parameters. To recognize disease related proteins that could serve as potential biomarkers is only feasible by investigating a non realizable large number of patients. Furthermore, plasma proteomics comprises enormous technical hurdles for quantitative analysis. High reproducibility of blood sampling in clinical routine is hard to achieve. Quantitative proteome analysis has to struggle with the complexity of millions of protein species comprising typical plasma proteins, cellular leakage proteins and antibodies and concentration differences of more than 1011 between high and low abundant proteins. Therefore, no successful quantitative and comprehensive plasma proteome analysis is reported so far. A novel proteomics strategy is proposed for biomarker discovery in plasma. Instead of comparing the plasma proteome of different individuals it is recommended to analyze the proteomes of different time points of a single individual during the development of a disease. This strategy is realized by the use of plasma of the Bavarian Red Cross Blood Bank, were three million samples are stored under standardized conditions. To achieve reliable data the isotope coded protein labelling proteomics technology was used.

  7. Multivariate Protein Signatures of Pre-Clinical Alzheimer's Disease in the Alzheimer's Disease Neuroimaging Initiative (ADNI) Plasma Proteome Dataset

    PubMed Central

    Johnstone, Daniel; Milward, Elizabeth A.; Berretta, Regina; Moscato, Pablo

    2012-01-01

    Background Recent Alzheimer's disease (AD) research has focused on finding biomarkers to identify disease at the pre-clinical stage of mild cognitive impairment (MCI), allowing treatment to be initiated before irreversible damage occurs. Many studies have examined brain imaging or cerebrospinal fluid but there is also growing interest in blood biomarkers. The Alzheimer's Disease Neuroimaging Initiative (ADNI) has generated data on 190 plasma analytes in 566 individuals with MCI, AD or normal cognition. We conducted independent analyses of this dataset to identify plasma protein signatures predicting pre-clinical AD. Methods and Findings We focused on identifying signatures that discriminate cognitively normal controls (n = 54) from individuals with MCI who subsequently progress to AD (n = 163). Based on p value, apolipoprotein E (APOE) showed the strongest difference between these groups (p = 2.3×10−13). We applied a multivariate approach based on combinatorial optimization ((α,β)-k Feature Set Selection), which retains information about individual participants and maintains the context of interrelationships between different analytes, to identify the optimal set of analytes (signature) to discriminate these two groups. We identified 11-analyte signatures achieving values of sensitivity and specificity between 65% and 86% for both MCI and AD groups, depending on whether APOE was included and other factors. Classification accuracy was improved by considering “meta-features,” representing the difference in relative abundance of two analytes, with an 8-meta-feature signature consistently achieving sensitivity and specificity both over 85%. Generating signatures based on longitudinal rather than cross-sectional data further improved classification accuracy, returning sensitivities and specificities of approximately 90%. Conclusions Applying these novel analysis approaches to the powerful and well-characterized ADNI dataset has identified sets of

  8. Comparative proteome analysis of cryopreserved flagella and head plasma membrane proteins from sea bream spermatozoa: effect of antifreeze proteins.

    PubMed

    Zilli, Loredana; Beirão, José; Schiavone, Roberta; Herraez, Maria Paz; Gnoni, Antonio; Vilella, Sebastiano

    2014-01-01

    Cryopreservation induces injuries to fish spermatozoa that in turn affect sperm quality in terms of fertilization ability, motility, DNA and protein integrity and larval survival. To reduce the loss of sperm quality due to freezing-thawing, it is necessary to improve these procedures. In the present study we investigated the ability of two antifreeze proteins (AFPI and AFPIII) to reduce the loss of quality of sea bream spermatozoa due to cryopreservation. To do so, we compared viability, motility, straight-line velocity and curvilinear velocity of fresh and (AFPs)-cryopreserved spermatozoa. AFPIII addition to cryopreservation medium improved viability, motility and straight-line velocity with respect to DMSO or DMSO plus AFPI. To clarify the molecular mechanism(s) underlying these findings, the protein profile of two different cryopreserved sperm domains, flagella and head plasma membranes, was analysed. The protein profiles differed between fresh and frozen-thawed semen and results of the image analysis demonstrated that, after cryopreservation, out of 270 proteins 12 were decreased and 7 were increased in isolated flagella, and out of 150 proteins 6 showed a significant decrease and 4 showed a significant increase in head membranes. Mass spectrometry analysis identified 6 proteins (4 from isolated flagella and 2 present both in flagella and head plasma membranes) within the protein spots affected by the freezing-thawing procedure. 3 out of 4 proteins from isolated flagella were involved in the sperm bioenergetic system. Our results indicate that the ability of AFPIII to protect sea bream sperm quality can be, at least in part, ascribed to reducing changes in the sperm protein profile occurring during the freezing-thawing procedure. Our results clearly demonstrated that AFPIII addition to cryopreservation medium improved the protection against freezing respect to DMSO or DMSO plus AFPI. In addition we propose specific proteins of spermatozoa as markers related to

  9. Comparative Proteome Analysis of Cryopreserved Flagella and Head Plasma Membrane Proteins from Sea Bream Spermatozoa: Effect of Antifreeze Proteins

    PubMed Central

    Zilli, Loredana; Beirão, José; Schiavone, Roberta; Herraez, Maria Paz; Gnoni, Antonio; Vilella, Sebastiano

    2014-01-01

    Cryopreservation induces injuries to fish spermatozoa that in turn affect sperm quality in terms of fertilization ability, motility, DNA and protein integrity and larval survival. To reduce the loss of sperm quality due to freezing-thawing, it is necessary to improve these procedures. In the present study we investigated the ability of two antifreeze proteins (AFPI and AFPIII) to reduce the loss of quality of sea bream spermatozoa due to cryopreservation. To do so, we compared viability, motility, straight-line velocity and curvilinear velocity of fresh and (AFPs)-cryopreserved spermatozoa. AFPIII addition to cryopreservation medium improved viability, motility and straight-line velocity with respect to DMSO or DMSO plus AFPI. To clarify the molecular mechanism(s) underlying these findings, the protein profile of two different cryopreserved sperm domains, flagella and head plasma membranes, was analysed. The protein profiles differed between fresh and frozen-thawed semen and results of the image analysis demonstrated that, after cryopreservation, out of 270 proteins 12 were decreased and 7 were increased in isolated flagella, and out of 150 proteins 6 showed a significant decrease and 4 showed a significant increase in head membranes. Mass spectrometry analysis identified 6 proteins (4 from isolated flagella and 2 present both in flagella and head plasma membranes) within the protein spots affected by the freezing-thawing procedure. 3 out of 4 proteins from isolated flagella were involved in the sperm bioenergetic system. Our results indicate that the ability of AFPIII to protect sea bream sperm quality can be, at least in part, ascribed to reducing changes in the sperm protein profile occurring during the freezing-thawing procedure. Our results clearly demonstrated that AFPIII addition to cryopreservation medium improved the protection against freezing respect to DMSO or DMSO plus AFPI. In addition we propose specific proteins of spermatozoa as markers related to

  10. Comparative proteomics of root plasma membrane proteins reveals the involvement of calcium signalling in NaCl-facilitated nitrate uptake in Salicornia europaea.

    PubMed

    Nie, Lingling; Feng, Juanjuan; Fan, Pengxiang; Chen, Xianyang; Guo, Jie; Lv, Sulian; Bao, Hexigeduleng; Jia, Weitao; Tai, Fang; Jiang, Ping; Wang, Jinhui; Li, Yinxin

    2015-08-01

    Improving crop nitrogen (N) use efficiency under salinity is essential for the development of sustainable agriculture in marginal lands. Salicornia europaea is a succulent euhalophyte that can survive under high salinity and N-deficient habitat conditions, implying that a special N assimilation mechanism may exist in this plant. In this study, phenotypic and physiological changes of S. europaea were investigated under different nitrate and NaCl levels. The results showed that NaCl had a synergetic effect with nitrate on the growth of S. europaea. In addition, the shoot nitrate concentration and nitrate uptake rate of S. europaea were increased by NaCl treatment under both low N and high N conditions, suggesting that nitrate uptake in S. europaea was NaCl facilitated. Comparative proteomic analysis of root plasma membrane (PM) proteins revealed 81 proteins, whose abundance changed significantly in response to NaCl and nitrate. These proteins are involved in metabolism, cell signalling, transport, protein folding, membrane trafficking, and cell structure. Among them, eight proteins were calcium signalling components, and the accumulation of seven of the above-mentioned proteins was significantly elevated by NaCl treatment. Furthermore, cytosolic Ca(2+) concentration ([Ca(2+)]cyt) was significantly elevated in S. europaea under NaCl treatment. The application of the Ca(2+) channel blocker LaCl3 not only caused a decrease in nitrate uptake rate, but also attenuated the promoting effects of NaCl on nitrate uptake rates. Based on these results, a possible regulatory network of NaCl-facilitated nitrate uptake in S. europaea focusing on the involvement of Ca(2+) signalling was proposed. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  11. Kidney proteome changes provide evidence for a dynamic metabolism and regional redistribution of plasma proteins during torpor-arousal cycles of hibernation

    PubMed Central

    Jani, Alkesh; Orlicky, David J.; Karimpour-Fard, Anis; Epperson, L. Elaine; Russell, Rae L.; Hunter, Lawrence E.

    2012-01-01

    Hibernating ground squirrels maintain homeostasis despite extreme physiological challenges. In winter, these circannual hibernators fast for months while cycling between prolonged periods of low blood flow and body temperature, known as torpor, and short interbout arousals (IBA), where more typical mammalian parameters are rapidly restored. Here we examined the kidney proteome for changes that support the dramatically different physiological demands of the hibernator's year. We identified proteins in 150 two-dimensional gel spots that altered by at least 1.5-fold using liquid chromatography and tandem mass spectrometry. These data successfully classified individuals by physiological state and revealed three dynamic patterns of relative protein abundance that dominated the hibernating kidney: 1) a large group of proteins generally involved with capturing and storing energy were most abundant in summer; 2) a select subset of these also increased during each arousal from torpor; and 3) 14 spots increased in torpor and early arousal were enriched for plasma proteins that enter cells via the endocytic pathway. Immunohistochemistry identified α2-macroglobulin and albumin in kidney blood vessels during late torpor and early arousal; both exhibited regional heterogeneity consistent with highly localized control of blood flow in the glomeruli. Furthermore, albumin, but not α2-macroglobulin, was detected in the proximal tubules during torpor and early arousal but not in IBA or summer animals. Taken together, our findings indicate that normal glomerular filtration barriers remain intact throughout torpor-arousal cycles but endocytosis, and hence renal function, is compromised at low body temperature during torpor and then recovers with rewarming during arousal. PMID:22643061

  12. Interpretation of plasma PTH concentrations according to 25OHD status, gender, age, weight status, and calcium intake: importance of the reference values.

    PubMed

    Touvier, Mathilde; Deschasaux, Mélanie; Montourcy, Marion; Sutton, Angela; Charnaux, Nathalie; Kesse-Guyot, Emmanuelle; Fezeu, Léopold K; Latino-Martel, Paule; Druesne-Pecollo, Nathalie; Malvy, Denis; Galan, Pilar; Hercberg, Serge; Ezzedine, Khaled; Souberbielle, Jean-Claude

    2014-04-01

    Reference values for plasma PTH assessment were generally established on small samples of apparently healthy subjects, without considering their 25-hydroxyvitamin D (25OHD) status or other potential modifiers of PTH concentration. Our objective was to assess ranges of plasma PTH concentration in a large sample of adults, stratifying by 25OHD status, age, gender, weight status, and calcium intake. This cross-sectional survey is based on 1824 middle-aged Caucasian adults from the Supplémentation en Vitamines et Minéraux Antioxydants study (1994). Plasma PTH and 25OHD concentrations were measured by an electrochemoluminescent immunoassay. Extreme percentiles of plasma PTH concentrations were assessed specifically in subjects who had plasmatic values of 25OHD of 20 ng/mL or greater and 30 ng/mL or greater. Among subjects with 25OHD status of 20 ng/mL or greater, the 97.5th percentile of plasma PTH concentration was 45.5 ng/L. By using this value as a reference, 5% of the subjects with plasma 25OHD less than 20 nmol/L had a high plasma PTH level, reflecting secondary hyperparathyroidism. Among vitamin D-replete subjects (25OHD status of 20 ng/mL or greater), the 97.5th percentile of plasma PTH was higher in overweight/obese subjects (51.9 vs 43.5 ng/L among normal weight subjects). The reference value for plasma PTH defined in this vitamin D-replete population was far below the value currently provided by the manufacturer (65 ng/L) and varied according to overweight status. These results may contribute to improve the diagnosis of primary and secondary hyperparathyroidism and subsequent therapeutic indication.

  13. Plant proteome analysis: a 2006 update.

    PubMed

    Jorrín, Jesús V; Maldonado, Ana M; Castillejo, Ma Angeles

    2007-08-01

    This 2006 'Plant Proteomics Update' is a continuation of the two previously published in 'Proteomics' by 2004 (Canovas et al., Proteomics 2004, 4, 285-298) and 2006 (Rossignol et al., Proteomics 2006, 6, 5529-5548) and it aims to bring up-to-date the contribution of proteomics to plant biology on the basis of the original research papers published throughout 2006, with references to those appearing last year. According to the published papers and topics addressed, we can conclude that, as observed for the three previous years, there has been a quantitative, but not qualitative leap in plant proteomics. The full potential of proteomics is far from being exploited in plant biology research, especially if compared to other organisms, mainly yeast and humans, and a number of challenges, mainly technological, remain to be tackled. The original papers published last year numbered nearly 100 and deal with the proteome of at least 26 plant species, with a high percentage for Arabidopsis thaliana (28) and rice (11). Scientific objectives ranged from proteomic analysis of organs/tissues/cell suspensions (57) or subcellular fractions (29), to the study of plant development (12), the effect of hormones and signalling molecules (8) and response to symbionts (4) and stresses (27). A small number of contributions have covered PTMs (8) and protein interactions (4). 2-DE (specifically IEF-SDS-PAGE) coupled to MS still constitutes the almost unique platform utilized in plant proteome analysis. The application of gel-free protein separation methods and 'second generation' proteomic techniques such as multidimensional protein identification technology (MudPIT), and those for quantitative proteomics including DIGE, isotope-coded affinity tags (ICAT), iTRAQ and stable isotope labelling by amino acids in cell culture (SILAC) still remains anecdotal. This review is divided into seven sections: Introduction, Methodology, Subcellular proteomes, Development, Responses to biotic and abiotic

  14. Blood folate status and expression of proteins involved in immune function, inflammation, and coagulation: biochemical and proteomic changes in the plasma of humans in response to long-term synthetic folic acid supplementation.

    PubMed

    Duthie, Susan J; Horgan, Graham; de Roos, Baukje; Rucklidge, Garry; Reid, Martin; Duncan, Gary; Pirie, Lynn; Basten, Graham P; Powers, Hilary J

    2010-04-05

    We used plasma proteomics to identify human proteins responsive to folate status. Plasma was collected from subjects treated with placebo or 1.2 mg of folic acid daily for 12 weeks in a randomized controlled trial. Homocysteine and folate were measured by immunoassay and uracil misincorporation by electrophoresis. The plasma proteome was assessed by 2-D gel electrophoresis, and proteins were identified by LC MS/MS. 5-methylTHF increased 5-fold (P = 0.000003) in response to intervention. Red cell folate doubled (P = 0.013), and lymphocyte folate increased 44% (P = 0.0001). Hcy and uracil dropped 22% (P = 0.0005) and 25% (P = 0.05), respectively. ApoE A-1, alpha-1-antichymotrypsin, antithrombin, and serum amyloid P were downregulated, while albumin, IgM C, and complement C3 were upregulated (P < 0.05). More than 60 proteins were significantly associated with folate pre- and postintervention (P < 0.01). These were categorized into metabolic pathways related to complement fixation (e.g., C1, C3, C4, Factor H, Factor 1, Factor B, clusterin), coagulation (e.g., antithrombin, alpha-1-antitrypsin, kininogen) and mineral transport (e.g., transthyretin, haptoglobin, ceruloplasmin). Low folate status pre- and post-treatment were associated with lower levels of proteins involved in activation and regulation of immune function and coagulation. Supplementation with synthetic folic acid increased expression of these proteins but did not substantially disrupt the balance of these pathways.

  15. Immunocamouflage of latex surfaces by grafted methoxypoly(ethylene glycol) (mPEG): proteomic analysis of plasma protein adsorption.

    PubMed

    Le, Yevgeniya; Li, Li; Wang, DunCheng; Scott, Mark D

    2012-03-01

    Grafting of methoxypoly(ethylene glycol) (mPEG) to cells and biomaterials is a promising non-pharmacological immunomodulation technology. However, due to the labile nature of cells, surface-plasma interactions are poorly understood; hence, a latex bead model was studied. PEGylation of beads resulted in a density and molecular weight dependent decrease in total adsorbed protein with a net reduction from (159.9±6.4) ng cm(-2) on bare latex to (18.4±0.8) and (52.3±5.3) ng cm(-2) on PEGylated beads (1 mmol L(-1) of 2 or 20 kD SCmPEG, respectively). SDS-PAGE and iTRAQ-MS analysis revealed differential compositions of the adsorbed protein layer on the PEGylated latex with a significant reduction in the compositional abundance of proteins involved in immune system activation. Thus, the biological efficacy of immunocamouflaged cells and materials is mediated by both biophysical obfuscation of antigens and reduced surface-macromolecule interactions.

  16. Nanoscale Proteomics

    SciTech Connect

    Shen, Yufeng; Tolic, Nikola; Masselon, Christophe D.; Pasa-Tolic, Liljiana; Camp, David G.; Anderson, Gordon A.; Smith, Richard D.; Lipton, Mary S.

    2004-02-01

    This paper describes efforts to develop a liquid chromatography (LC)/mass spectrometry (MS) technology for ultra-sensitive proteomics studies, i.e. nanoscale proteomics. The approach combines high-efficiency nano-scale LC with advanced MS, including high sensitivity and high resolution Fourier transform ion cyclotron resonance (FTICR) MS, to perform both single-stage MS and tandem MS (MS/MS) proteomic analyses. The technology developed enables large-scale protein identification from nanogram size proteomic samples and characterization of more abundant proteins from sub-picogram size complex samples. Protein identification in such studies using MS is feasible from <75 zeptomole of a protein, and the average proteome measurement throughput is >200 proteins/h and ~3 h/sample. Higher throughput (>1000 proteins/h) and more sensitive detection limits can be obtained using a “accurate mass and time” tag approach developed at our laboratory. These capabilities lay the foundation for studies from single or limited numbers of cells.

  17. Analysis of the Oryza sativa plasma membrane proteome using combined protein and peptide fractionation approaches in conjunction with mass spectrometry.

    PubMed

    Natera, Siria H A; Ford, Kristina L; Cassin, Andrew M; Patterson, John H; Newbigin, Edward J; Bacic, Antony

    2008-03-01

    To identify integral and peripheral plasma membrane (PM) proteins from Oryza sativa (rice), highly enriched PM fractions from rice suspension cultured cells were analyzed using two complementary approaches. The PM was enriched using aqueous two-phase partitioning and high pH carbonate washing to remove soluble, contaminating proteins and characterized using enzymatic and immunological analyses. Proteins from the carbonate-washed PM (WPM) were analyzed by either one-dimensional gel electrophoresis (1D-SDS-PAGE) followed by tryptic proteolysis or proteolysis followed by strong cation exchange liquid chromatography (LC) with subsequent analysis of the tryptic peptides by LC-MS/MS (termed Gel-LC-MS/MS and 2D-LC-MS/MS, respectively). Combining the results of these two approaches, 438 proteins were identified on the basis of two or more matching peptides, and a further 367 proteins were identified on the basis of single peptide matches after data analysis with two independent search algorithms. Of these 805 proteins, 350 were predicted to be PM or PM-associated proteins. Four hundred and twenty-five proteins (53%) were predicted to be integrally associated with a membrane, via either one or many (up to 16) transmembrane domains, a GPI-anchor, or membrane-spanning beta-barrels. Approximately 80% of the 805 identified proteins were assigned a predicted function, based on similarity to proteins of known function or the presence of functional domains. Proteins involved in PM-related activities such as signaling (21% of the 805 proteins), transporters and ATPases (14%), and cellular trafficking (8%), such as via vesicles involved in endo- and exocytosis, were identified. Proteins that are involved in cell wall biosynthesis were also identified (5%) and included three cellulose synthase (CESA) proteins, a cellulose synthase-like D (CSLD) protein, cellulases, and several callose synthases. Approximately 20% of the proteins identified in this study remained functionally

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

    PubMed Central

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

    2013-01-01

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

  19. Reference Intervals of Mineral Elements in Plasma of Anesthetized Free-Ranging Adult Females of South American Sea Lion, Otaria flavescens.

    PubMed

    Polizzi, Paula S; Boudet, L N Chiodi; de León, A Ponce; Quiroga, M; Rodríguez, D H; Atkinson, S; Gerpe, M S

    2016-12-01

    Levels of zinc (Zn), copper (Cu), potassium (K), calcium (Ca), magnesium (Mg), and phosphorus (P) in plasma of Otaria flavescens females (n = 29) were evaluated. Reference intervals were established for each element, being the first report for this species.

  20. Background | Office of Cancer Clinical Proteomics Research

    Cancer.gov

    The term "proteomics" refers to a large-scale comprehensive study of a specific proteome resulting from its genome, including abundances of proteins, their variations and modifications, and interacting partners and networks in order to understand cellular processes involved.  Similarly, “Cancer proteomics” refers to comprehensive analyses of proteins and their derivatives translated from a specific cancer genome using a human biospecimen or a preclinical model (e.g., cultured cell or animal model).

  1. Proteomic analysis of urine exosomes by multidimensional protein identification technology (MudPIT).

    PubMed

    Wang, Zhen; Hill, Salisha; Luther, James M; Hachey, David L; Schey, Kevin L

    2012-01-01

    Exosomes are membrane vesicles that are secreted by cells upon fusion of multivesicular bodies with the plasma membrane. Exosomal proteomics has emerged as a powerful approach to understand the molecular composition of exosomes and has potential to accelerate biomarker discovery. Different proteomic analysis methods have been previously employed to establish several exosome protein databases. In this study, TFE solution-phase digestion was compared with in-gel digestion and found to yield similar results. Proteomic analysis of urinary exosomes was performed by multidimensional protein identification technology (MudPIT) after TFE digestion. Nearly, 3280 proteins were identified from nine human urine samples with 31% overlap among nine samples. Gene ontology (GO) analysis, coupled with detection of all of the members of ESCRT machinery complex, supports the multivesicular origin of these particles. These results significantly expand the existing database of urinary exosome proteins. Our results also indicate that more than 1000 proteins can be detected from exosomes prepared from as little as 25 mL of urine. This study provides the largest set of proteins present in human urinary exosome proteomes, provides a valuable reference for future studies, and provides methods that can be applied to exosomal proteomic analysis from other tissue sources.

  2. What is Proteomics? - Office of Cancer Clinical Proteomics Research

    Cancer.gov

    The term "proteome" refers to the entire complement of proteins, including the modifications made to a particular set of proteins, produced by an organism or a cellular system. This will vary with time and distinct requirements, such as stresses, that a cell or organism undergoes.

  3. What is Proteomics? - Office of Cancer Clinical Proteomics Research

    Cancer.gov

    The term "proteome" refers to the entire complement of proteins, including the modifications made to a particular set of proteins, produced by an organism or a cellular system. This will vary with time and distinct requirements, such as stresses, that a cell or organism undergoes.

  4. Plasma erythropoietin assay by a fetal mouse liver cell culture method with special reference to effective elimination of erythroid colony inhibitor(s) in plasma.

    PubMed

    Sakata, S; Enoki, Y; Nakatani, A; Kohzuki, H; Ohga, Y; Shimizu, S

    1987-03-01

    Methods for the elimination of an inhibitor(s) of erythroid colony formation from plasma were examined in an attempt to measure genuine plasma erythropoietin (Epo) activities with an erythroid colony-forming assay using fetal mouse liver cells. Acid-boiling-chloroform (ABC) treatment was concluded to be the best method because the plasma thus treated stimulated colony formation most and contained the least protein. The dose-response curve for the plasma was parallel to that for the standard Epo preparation. The "erythroid colony-stimulating activity" in the plasma was completely additive to that in the standard Epo, and appeared to be a relatively heat-stable and acid protein with an isoelectric point lower than 5.0. These results suggest that the activity in the plasma is identical to that in the standard Epo. Stability of the plasma Epo activity was dependent on storage temperature and enhanced by adding 1% bovine serum albumin (BSA). Average Epo titers for normal adult, full-term cord, and murine plasmas, all ABC-treated and with 1% BSA added, were 192.4, 184.5, and 150.6 mU/ml, respectively. These values were much higher than those measured by the in vivo standard polycythemic mouse assay.

  5. Proteomics studies of pancreatic cancer

    PubMed Central

    Chen, Ru; Pan, Sheng; Aebersold, Ruedi; Brentnall, Teresa A.

    2008-01-01

    Pancreatic cancer is the fourth leading cause of cancer death in the United States, with 4% survival 5 years after diagnosis. Biomarkers are desperately needed to improve earlier, more curable cancer diagnosis and to develop new effective therapeutic targets. The development of quantitative proteomics technologies in recent years offers great promise for understanding the complex molecular events of tumorigenesis at the protein level, and has stimulated great interest in applying the technology for pancreatic cancer studies. Proteomic studies of pancreatic tissues, juice, serum/plasma, and cell lines have recently attempted to identify differentially expressed proteins in pancreatic cancer to dissect the abnormal signaling pathways underlying oncogenesis, and to detect new biomarkers. It can be expected that the continuing evolution of proteomics technology with better resolution and sensitivity will greatly enhance our capability in combating pancreatic cancer. PMID:18633454

  6. Evaluation of plasma antioxidant activity in rats given excess EGCg with reference to endogenous antioxidants concentrations and assay methods.

    PubMed

    Yokotani, Kaori; Umegaki, Keizo

    2017-02-01

    The contribution of (-)-epigallocatechin gallate (EGCg) intake to in vivo antioxidant activity is unclear, even with respect to plasma. In this study, we examined how administration of EGCg contributes to plasma antioxidant activity, relative to its concentration, endogenous antioxidants, and assay methods, namely oxygen radical absorbance capacity (ORAC) and ferric reducing/antioxidant power (FRAP). Administration of EGCg (500 mg/kg) to rats increased plasma EGCg (4μmol/L as free form) and ascorbic acid (1.7-fold), as well as ORAC (1.2-fold) and FRAP (3-fold) values. The increase in plasma ascorbic acid following EGCg administration was accompanied by its relocation from the adrenal glands and lymphocytes into plasma, and was related to the increase in FRAP. Plasma deproteinization and assays in plasma model solutions revealed that protein levels significantly contributed to ORAC values, where <3 μmol/L EGCg in the presence of protein exhibited minimal antioxidant activity, as measured by both FRAP and ORAC. As the concentration of plasma ascorbic acid was not influenced by deproteinization, differences in FRAP values with and without deproteinization were estimated to determine the contribution of enhanced ascorbic acid attributable to EGCg administration. These results will help to understand the points that should be considered when evaluating EGCg antioxidant activity in plasma.

  7. Quantification of drugs in plasma without primary reference standards by liquid chromatography-chemiluminescence nitrogen detection: application to tramadol metabolite ratios.

    PubMed

    Ojanperä, Suvi; Rasanen, Ilpo; Sistonen, Johanna; Pelander, Anna; Vuori, Erkki; Ojanperä, Ilkka

    2007-08-01

    Lack of availability of reference standards for drug metabolites, newly released drugs, and illicit drugs hinders the analysis of these substances in biologic samples. To counter this problem, an approach is presented here for quantitative drug analysis in plasma without primary reference standards by liquid chromatography-chemiluminescence nitrogen detection (LC-CLND). To demonstrate the feasibility of the method, metabolic ratios of the opioid drug tramadol were determined in the setting of a pharmacogenetic study. Four volunteers were given a single 100-mg oral dose of tramadol, and a blood sample was collected from each subject 1 hour later. Tramadol, O-desmethyltramadol, and nortramadol were determined in plasma by LC-CLND without reference standards and by a gas chromatography-mass spectrometry reference method. In contrast to previous CLND studies lacking an extraction step, a liquid-liquid extraction system was created for 5-mL plasma samples using n-butyl chloride-isopropyl alcohol (98 + 2) at pH 10. Extraction recovery estimation was based on model compounds chosen according to their similar physicochemical characteristics (retention time, pKa, logD). Instrument calibration was performed with a single secondary standard (caffeine) using the equimolar response of the detector to nitrogen. The mean differences between the results of the LC-CLND and gas chromatography-mass spectrometry methods for tramadol, O-desmethyltramadol, and nortramadol were 8%, 32%, and 19%, respectively. The sensitivity of LC-CLND was sufficient for therapeutic concentrations of tramadol and metabolites. A good correlation was obtained between genotype, expressed by the number of functional genes, and the plasma metabolite ratios. This experiment suggests that a recovery-corrected LC-CLND analysis produces sufficiently accurate results to be useful in a clinical context, particularly in instances in which reference standards are not readily accessible.

  8. Consolidation of proteomics data in the Cancer Proteomics database.

    PubMed

    Arntzen, Magnus Ø; Boddie, Paul; Frick, Rahel; Koehler, Christian J; Thiede, Bernd

    2015-11-01

    Cancer is a class of diseases characterized by abnormal cell growth and one of the major reasons for human deaths. Proteins are involved in the molecular mechanisms leading to cancer, furthermore they are affected by anti-cancer drugs, and protein biomarkers can be used to diagnose certain cancer types. Therefore, it is important to explore the proteomics background of cancer. In this report, we developed the Cancer Proteomics database to re-interrogate published proteome studies investigating cancer. The database is divided in three sections related to cancer processes, cancer types, and anti-cancer drugs. Currently, the Cancer Proteomics database contains 9778 entries of 4118 proteins extracted from 143 scientific articles covering all three sections: cell death (cancer process), prostate cancer (cancer type) and platinum-based anti-cancer drugs including carboplatin, cisplatin, and oxaliplatin (anti-cancer drugs). The detailed information extracted from the literature includes basic information about the articles (e.g., PubMed ID, authors, journal name, publication year), information about the samples (type, study/reference, prognosis factor), and the proteomics workflow (Subcellular fractionation, protein, and peptide separation, mass spectrometry, quantification). Useful annotations such as hyperlinks to UniProt and PubMed were included. In addition, many filtering options were established as well as export functions. The database is freely available at http://cancerproteomics.uio.no.

  9. Plasma exogenous creatinine clearance in clinically healthy cats: comparison with urinary exogenous creatinine clearance, tentative reference intervals and indexation to bodyweight.

    PubMed

    Reynolds, B S; Massal, M R; Nguyen, P; Grégoire, L L; Périgaud, A E; Concordet, D; Biourge, V; Lefebvre, H P

    2014-10-01

    Glomerular filtration rate (GFR) is considered to be the best indicator of overall kidney function. The major objectives of this study were to compare plasma exogenous creatinine clearance (PECC) with a reference method, to establish reference intervals (RIs) for PECC and to assess the effects of indexation of GFR to bodyweight (BW) in cats. PECC was compared with urinary clearance of exogenous creatinine (UECC) in six clinically healthy domestic shorthair cats (experiment 1). Tentative RIs were determined according to current guidelines and the effects of indexation to BW and of covariables on GFR were assessed in 43 clinically healthy cats of various breeds (experiment 2). PECC was 15% higher than UECC (P <0.01), but the two estimates were strongly correlated (r(2)=0.97, P = 0.001). RIs for PECC were 6.4-21.3 mL/min or 1.2-4.9 mL/min/kg. The absolute (i.e. non-indexed) GFR value was not dependent on BW. Thus, indexation of GFR to BW in cats would not standardize the GFR value, but could introduce bias in clinical interpretation. Significant effects of breed, plasma protein concentration and plasma albumin concentration on GFR were demonstrated. Plasma concentrations of urea and creatinine, when assessed separately, were also weakly correlated with GFR in healthy cats. These combined findings contribute to a better understanding of renal function assessment in cats.

  10. Estimation of reference intervals of five endocannabinoids and endocannabinoid related compounds in human plasma by two dimensional-LC/MS/MS

    PubMed Central

    Fanelli, Flaminia; Di Lallo, Valentina D.; Belluomo, Ilaria; De Iasio, Rosaria; Baccini, Margherita; Casadio, Elena; Gasparini, Daniela Ibarra; Colavita, Michelangelo; Gambineri, Alessandra; Grossi, Gabriele; Vicennati, Valentina; Pasquali, Renato; Pagotto, Uberto

    2012-01-01

    The elucidation of the role of endocannabinoids in physiological and pathological conditions and the transferability of the importance of these mediators from basic evidence into clinical practice is still hampered by the indefiniteness of their circulating reference intervals. In this work, we developed and validated a two-dimensional LC/MS/MS method for the simultaneous measurement of plasma endocannabinoids and related compounds such as arachidonoyl-ethanolamide, palmitoyl-ethanolamide, and oleoyl-ethanolamide, belonging to the N-acyl-ethanolamide (NAE) family, and 2-arachidonoyl-glycerol and its inactive isomer 1-arachidonoyl-glycerol from the monoacyl-glycerol (MAG) family. We found that several pitfalls in the endocannabinoid measurement may occur, from blood withdrawal to plasma processing. Plasma extraction with toluene followed by on-line purification was chosen, allowing high-throughput and reliability. We estimated gender-specific reference intervals on 121 healthy normal weight subjects fulfilling rigorous anthropometric and hematic criteria. We observed no gender differences for NAEs, whereas significantly higher MAG levels were found in males compared with females. MAGs also significantly correlated with triglycerides. NAEs increased with age in females, and arachidonoyl-ethanolamide correlated with adiposity and metabolic parameters in females. This work paves the way to the establishment of definitive reference intervals for circulating endocannabinoids to help physicians move from the speculative research field into the clinical field. PMID:22172516

  11. Proteome | Office of Cancer Clinical Proteomics Research

    Cancer.gov

    A proteome is the entire complement of proteins, including modifications made to a particular set of proteins, produced by an organism or a cellular system. This will vary with time and distinct requirements such as growth conditions and stresses, and thus is highly dynamic and spatial. Proteomics is the study of the proteome.

  12. Plasma osmolality reference values in African grey parrots (Psittacus erithacus erithacus), Hispaniolan Amazon parrots (Amazona ventralis), and red-fronted macaws (Ara rubrogenys).

    PubMed

    Beaufrère, Hugues; Acierno, Mark; Mitchell, Mark; Guzman, David Sanchez-Migallon; Bryant, Heather; Tully, Thomas N

    2011-06-01

    Birds are routinely presented to veterinarians for dehydration. Success with these cases ultimately depends on providing replacement fluids and re-establishing fluid homeostasis. Few studies have been done to determine reference ranges for plasma osmolality in birds. The goals of this study were to determine reference values for plasma osmolality in 3 species of parrots and to provide recommendations on fluid selection for replacement therapy in these species. Blood samples were collected from 21 adult Hispaniolan Amazon parrots (Amazona ventralis), 21 Congo African grey parrots (Psittacus erithacus erithacus), and 9 red-fronted macaws (Ara rubrogenys), and were placed into lithium heparin containers. Plasma osmolality was measured in duplicate with a freezing point depression osmometer. Summary statistics were computed from the average values. Reference ranges, calculated by using the robust method, were 288-324, 308-345, and 223-369 mOsm/kg in African grey parrots, Hispaniolan Amazon parrots, and red-fronted macaws, respectively. The mean +/- SD values were 306 +/- 7, 327 +/- 7, and 304 +/- 18 mOsm/kg in African grey parrots, Hispaniolan Amazon parrots, and red-fronted macaws, respectively. Comparisons with osmolality values in mammals and values previously reported for psittacine bird species suggest that plasma osmolality is slightly higher in parrots than in mammals, species-specific differences exist, and differences between reported values occur. Overall, fluids with an osmolarity close to 300-320 mOsm/L, such as Normosol-R, Plasmalyte-R, Plasmalyte-A, and NaCl 0.9%, can be recommended in parrots for fluid replacement therapy when isotonic fluids are required.

  13. Proteomic Investigations into Hemodialysis Therapy

    PubMed Central

    Bonomini, Mario; Sirolli, Vittorio; Pieroni, Luisa; Felaco, Paolo; Amoroso, Luigi; Urbani, Andrea

    2015-01-01

    The retention of a number of solutes that may cause adverse biochemical/biological effects, called uremic toxins, characterizes uremic syndrome. Uremia therapy is based on renal replacement therapy, hemodialysis being the most commonly used modality. The membrane contained in the hemodialyzer represents the ultimate determinant of the success and quality of hemodialysis therapy. Membrane’s performance can be evaluated in terms of removal efficiency for unwanted solutes and excess fluid, and minimization of negative interactions between the membrane material and blood components that define the membrane’s bio(in)compatibility. Given the high concentration of plasma proteins and the complexity of structural functional relationships of this class of molecules, the performance of a membrane is highly influenced by its interaction with the plasma protein repertoire. Proteomic investigations have been increasingly applied to describe the protein uremic milieu, to compare the blood purification efficiency of different dialyzer membranes or different extracorporeal techniques, and to evaluate the adsorption of plasma proteins onto hemodialysis membranes. In this article, we aim to highlight investigations in the hemodialysis setting making use of recent developments in proteomic technologies. Examples are presented of why proteomics may be helpful to nephrology and may possibly affect future directions in renal research. PMID:26690416

  14. Selected reaction monitoring applied to proteomics.

    PubMed

    Gallien, Sebastien; Duriez, Elodie; Domon, Bruno

    2011-03-01

    Selected reaction monitoring (SRM) performed on triple quadrupole mass spectrometers has been the reference quantitative technique to analyze small molecules for several decades. It is now emerging in proteomics as the ideal tool to complement shotgun qualitative studies; targeted SRM quantitative analysis offers high selectivity, sensitivity and a wide dynamic range. However, SRM applied to proteomics presents singularities that distinguish it from small molecules analysis. This review is an overview of SRM technology and describes the specificities and the technical aspects of proteomics experiments. Ongoing developments aiming at increasing multiplexing capabilities of SRM are discussed; they dramatically improve its throughput and extend its field of application to directed or supervised discovery experiments.

  15. Accounting for population variation in targeted proteomics.

    PubMed

    Fujimoto, Grant M; Monroe, Matthew E; Rodriguez, Larissa; Wu, Chaochao; MacLean, Brendan; Smith, Richard D; MacCoss, Michael J; Payne, Samuel H

    2014-01-03

    Individual proteomes typically differ from the reference human proteome at ∼10,000 single amino acid variants. When viewed on the population scale, this individual variation results in a wide variety of protein sequences. In targeted proteomics experiments, such variability can confound accurate protein quantification. To assist researchers in identifying target peptides with high variability within the human population, we have created the Population Variation plug-in for Skyline, which provides easy access to the polymorphisms stored in dbSNP. Given a set of peptides, the tool reports minor allele frequency for common polymorphisms. We highlight the importance of considering genetic variation by applying the tool to public data sets.

  16. Accounting for Population Variation in Targeted Proteomics

    SciTech Connect

    Fujimoto, Grant M.; Monroe, Matthew E.; Rodriguez, Larissa; Wu, Chaochao; MacLean, Brendan; Smith, Richard D.; MacCoss, Michael J.; Payne, Samuel H.

    2014-01-03

    Individual proteomes typically differ from the reference human proteome at ~10,000 single amino acid variants. When viewed at the population scale, this individual variation results in a wide variety of protein sequences. In targeted proteomics experiments, such variability would confound accurate protein quantification. To facilitate researchers in identifying target peptides with high variability within the human population we have created the Population Variation plug-in for Skyline, which provides easy access to the polymorphisms stored in dbSNP. Given a set of peptides, the tool reports minor allele frequency for common polymorphisms. We highlight the importance of considering genetic variation by applying the tool to public datasets.

  17. Endothelial Cell Proteomic Response to Rickettsia conorii Infection Reveals Activation of the Janus Kinase (JAK)-Signal Transducer and Activator of Transcription (STAT)-Inferferon Stimulated Gene (ISG)15 Pathway and Reprogramming Plasma Membrane Integrin/Cadherin Signaling*

    PubMed Central

    Zhao, Yingxin; Valbuena, Gustavo; Walker, David H.; Gazi, Michal; Hidalgo, Marylin; DeSousa, Rita; Oteo, Jose Antonio; Goez, Yenny; Brasier, Allan R.

    2016-01-01

    Rickettsia conorii is the etiologic agent of Mediterranean spotted fever, a re-emerging infectious disease with significant mortality. This Gram-negative, obligately intracellular pathogen is transmitted via tick bites, resulting in disseminated vascular endothelial cell infection with vascular leakage. In the infected human, Rickettsia conorii infects endothelial cells, stimulating expression of cytokines and pro-coagulant factors. However, the integrated proteomic response of human endothelial cells to R. conorii infection is not known. In this study, we performed quantitative proteomic profiling of primary human umbilical vein endothelial cells (HUVECs) with established R conorii infection versus those stimulated with endotoxin (LPS) alone. We observed differential expression of 55 proteins in HUVEC whole cell lysates. Of these, we observed induction of signal transducer and activator of transcription (STAT)1, MX dynamin-like GTPase (MX1), and ISG15 ubiquitin-like modifier, indicating activation of the JAK-STAT signaling pathway occurs in R. conorii-infected HUVECs. The down-regulated proteins included those involved in the pyrimidine and arginine biosynthetic pathways. A highly specific biotinylated cross-linking enrichment protocol was performed to identify dysregulation of 11 integral plasma membrane proteins that included up-regulated expression of a sodium/potassium transporter and down-regulation of α-actin 1. Analysis of Golgi and soluble Golgi fractions identified up-regulated proteins involved in platelet-endothelial adhesion, phospholipase activity, and IFN activity. Thirty four rickettsial proteins were identified with high confidence in the Golgi, plasma membrane, or secreted protein fractions. The host proteins associated with rickettsial infections indicate activation of interferon-STAT signaling pathways; the disruption of cellular adhesion and alteration of antigen presentation pathways in response to rickettsial infections are distinct from

  18. Seed proteomics.

    PubMed

    Miernyk, Ján A; Hajduch, Martin

    2011-04-01

    Seeds comprise a protective covering, a small embryonic plant, and a nutrient-storage organ. Seeds are protein-rich, and have been the subject of many mass spectrometry-based analyses. Seed storage proteins (SSP), which are transient depots for reduced nitrogen, have been studied for decades by cell biologists, and many of the complicated aspects of their processing, assembly, and compartmentation are now well understood. Unfortunately, the abundance and complexity of the SSP requires that they be avoided or removed prior to gel-based analysis of non-SSP. While much of the extant data from MS-based proteomic analysis of seeds is descriptive, it has nevertheless provided a preliminary metabolic picture explaining much of their biology. Contemporary studies are moving more toward analysis of protein interactions and posttranslational modifications, and functions of metabolic networks. Many aspects of the biology of seeds make then an attractive platform for heterologous protein expression. Herein we present a broad review of the results from the proteomic studies of seeds, and speculate on a potential future research directions. Copyright © 2010 Elsevier B.V. All rights reserved.

  19. Mechanisms of referred visceral pain: uterine inflammation in the adult virgin rat results in neurogenic plasma extravasation in the skin.

    PubMed

    Wesselmann, U; Lai, J

    1997-12-01

    The purpose of this study was to investigate the mechanisms of referred pain observed in female patients with pain from the reproductive organs. We developed a model of inflammatory uterine pain in the rat. Inflammation of the uterus in rats pretreated with Evans Blue Dye resulted in dye extravasation in the skin over the abdomen, groin, lower back, thighs, perineal area and proximal tail, thus providing for the first time evidence for the trophic changes observed in the area of referred visceral pain in an animal model of uterine pain. The neuronal pathways mediating the observed dye extravasation in the skin after uterine inflammation may include dichotomizing afferent fibers, afferent-afferent interactions via a spinal cord pathway or a sympathetic reflex. This model will allow to gain further insight into the mechanisms of referred pain and the trophic changes observed in the area of referred pain in visceral disease.

  20. Proteomic Biomarkers of Atherosclerosis

    PubMed Central

    Vivanco, F.; Padial, L.R.; Darde, V.M.; de la Cuesta, F.; Alvarez-Llamas, G.; Diaz-Prieto, Natacha; Barderas, M.G.

    2008-01-01

    Summary Biomarkers provide a powerful approach to understanding the spectrum of cardiovascular diseases. They have application in screening, diagnostic, prognostication, prediction of recurrences and monitoring of therapy. The “omics” tool are becoming very useful in the development of new biomarkers in cardiovascular diseases. Among them, proteomics is especially fitted to look for new proteins in health and disease and is playing a significant role in the development of new diagnostic tools in cardiovascular diagnosis and prognosis. This review provides an overview of progress in applying proteomics to atherosclerosis. First, we describe novel proteins identified analysing atherosclerotic plaques directly. Careful analysis of proteins within the atherosclerotic vascular tissue can provide a repertoire of proteins involved in vascular remodelling and atherogenesis. Second, we discuss recent data concerning proteins secreted by atherosclerotic plaques. The definition of the atheroma plaque secretome resides in that proteins secreted by arteries can be very good candidates of novel biomarkers. Finally we describe proteins that have been differentially expressed (versus controls) by individual cells which constitute atheroma plaques (endothelial cells, vascular smooth muscle cells, macrophages and foam cells) as well as by circulating cells (monocytes, platelets) or novel biomarkers present in plasma. PMID:19578499

  1. A WHO reference reagent to standardize haemagglutination testing for anti-A and anti-B in serum and plasma: international collaborative study to evaluate a candidate preparation.

    PubMed

    Thorpe, S J; Fox, B; Sharp, G; White, J; Milkins, C

    2016-08-01

    The aim of the study was to evaluate a lyophilized serum preparation, 14/300, for its suitability to serve as a World Health Organization (WHO) Reference Reagent to standardize and control haemagglutination titrations for anti-A and anti-B in serum and plasma, in an international collaborative study. Serum preparation 14/300 and two plasma-based reserve preparations, 14/304 (high titre anti-A) and 14/208 (high titre anti-B), were titrated by 24 laboratories in 13 countries using direct (DRT) and indirect (IAT) haemagglutination techniques. There was eightfold to 64-fold variation in reported titres per preparation and method across laboratories, that is, titres extended over 4-7 dilutions, although intralaboratory variability was generally good, with over 90% of replicate titres within a twofold range. There was a reduction in interlaboratory variability when titres of the reserve preparations were adjusted relative to those of the candidate Reference Reagent. The establishment of 14/300 as a WHO Reference Reagent for high titre anti-A and anti-B in serum, with nominal anti-A and anti-B titres of 128 for DRT, and nominal anti-A and anti-B titres of 256 for IAT, will facilitate global standardization of haemagglutination titrations for anti-A and anti-B in patient samples and blood components. © 2016 Crown copyright. Vox Sanguinis © 2016 International Society of Blood Transfusion.

  2. Reference intervals for select hematologic and plasma biochemical analytes of wild Lake Sturgeon (Acipenser fulvescens) from the St. Lawrence River in New York.

    PubMed

    DiVincenti, Louis; Wyatt, Jeff; Priest, Heather; Dittman, Dawn; Klindt, Rodger; Gordon, David; Preston, Andrew; Smith, Thomas; Bowman, Colby

    2013-03-01

    Lake Sturgeon (Acipenser fulvescens) is a long-lived freshwater fish distributed throughout the Great Lakes region and is a threatened species in New York State. The species' unique life cycle makes it useful as an indicator of ecosystem health, and efforts to repatriate Lake Sturgeon to their historic range are underway. However, comprehensive hematologic and biochemical reference intervals that would be valuable for assessing the health status of individual animals have not been reported. The objective of this study was to determine normal hematologic and plasma biochemical reference intervals for healthy wild Lake Sturgeon. Blood samples were collected from 52 wild Lake Sturgeon caught in gill nets in the St. Lawrence River. Heparinized whole blood and plasma samples were analyzed using standard techniques. Reference intervals were calculated using the robust method following elimination of outliers and Box-Cox transformation of data. Hematologic reference intervals were as follows: PCV 17-38%, estimated WBC count 2740-23,150/μL, neutrophils 193-6121/μL, eosinophils 0-558/μL, other granulocytes/heterophils 0-488/μL, lymphocytes 1447-14,044/μL, and monocytes 55-1684/μL. Plasma biochemical reference intervals were as follows: aspartate aminotransferase 333-1746 U/L, calcium 1.85-2.80 mmol/L, chloride 95-123 mmol/L, creatine kinase 776-35,536 U/L, glucose 2.94-14.76 mmol/L, glutamate dehydrogenase 6-30 U/L, phosphate 2.03-5.81 mmol/L, potassium 2.34-4.24 mmol/L, sodium 122.9-151.1 mmol/L, total protein 2.0-4.4 g/dL, triglycerides 1.07-5.12 mmol/L, and uric acid 1-251 μmol/L. Reference intervals reported here will be useful for health assessment of wild and repatriated Lake Sturgeon. © 2012 American Society for Veterinary Clinical Pathology.

  3. Hematology and Plasma Chemistry Reference Intervals for Mature Laboratory Pine Voles (Microtus pinetorum) as Determined by Using the Nonparametric Rank Percentile Method

    PubMed Central

    Harvey, Stephen B; Krimer, Paula M; Correa, Maria T; Hanes, Martha A

    2008-01-01

    Plasma biochemical and hematologic values are important parameters for assessing animal health and experimental results. Although normal reference values for many rodent species have been published, there is a dearth of similar information for the genus Microtus. In addition, most studies use a mean and standard deviation to establish reference intervals, but doing so is not the recommendation of the Clinical and Laboratory Standards Institute (formerly the National Committee on Clinical Laboratory Standards) or the International Federation of Clinical Chemistry and Laboratory Medicine. The purpose of this study was to establish normal reference parameters for plasma biochemistry and hematology in mature pine voles (Microtus pinetorum) by using the nonparametric rank percentile method as recommended by the 2 laboratory medicine organizations mentioned. Samples of cardiac blood from a closed colony of pine voles were collected at euthanasia and evaluated under rodent settings on 2 automated hematology analyzers from 2 different manufacturers and on the same type of automated biochemistry analyzer. There were no sex-associated clinically significant differences between the sexes; younger animals had a lower hematocrit, higher mean corpuscular volume, and lower mean corpuscular hemoglobin concentration than did older animals. Only platelet counts differed when comparing hematologic values from different analyzers. Relative to rats and mice, pine voles have a lower mean corpuscular volume and higher red blood cell count, higher blood urea nitrogen, much higher alanine aminotransferase, and lower glucose and phosphorous concentrations. Hematology and plasma biochemical results obtained in this study are considered representative for healthy adult laboratory pine voles under similar environmental conditions. PMID:18702449

  4. Hematology and plasma chemistry reference intervals for mature laboratory pine voles (Microtus pinetorum) as determined by using the nonparametric rank percentile method.

    PubMed

    Harvey, Stephen B; Krimer, Paula M; Correa, Maria T; Hanes, Martha A

    2008-07-01

    Plasma biochemical and hematologic values are important parameters for assessing animal health and experimental results. Although normal reference values for many rodent species have been published, there is a dearth of similar information for the genus Microtus. In addition, most studies use a mean and standard deviation to establish reference intervals, but doing so is not the recommendation of the Clinical and Laboratory Standards Institute (formerly the National Committee on Clinical Laboratory Standards) or the International Federation of Clinical Chemistry and Laboratory Medicine. The purpose of this study was to establish normal reference parameters for plasma biochemistry and hematology in mature pine voles (Microtus pinetorum) by using the nonparametric rank percentile method as recommended by the 2 laboratory medicine organizations mentioned. Samples of cardiac blood from a closed colony of pine voles were collected at euthanasia and evaluated under rodent settings on 2 automated hematology analyzers from 2 different manufacturers and on the same type of automated biochemistry analyzer. There were no sex-associated clinically significant differences between the sexes; younger animals had a lower hematocrit, higher mean corpuscular volume, and lower mean corpuscular hemoglobin concentration than did older animals. Only platelet counts differed when comparing hematologic values from different analyzers. Relative to rats and mice, pine voles have a lower mean corpuscular volume and higher red blood cell count, higher blood urea nitrogen, much higher alanine aminotransferase, and lower glucose and phosphorous concentrations. Hematology and plasma biochemical results obtained in this study are considered representative for healthy adult laboratory pine voles under similar environmental conditions.

  5. Hematologic and plasma biochemical reference intervals for Morelet's crocodiles (Crocodylus moreletii) in the northern wetlands of Campeche, Mexico.

    PubMed

    Padilla, Sergio E; Weber, Manuel; Jacobson, Elliott R

    2011-07-01

    Health surveys and hematologic and plasma biochemical analyses were conducted in 52 free-ranging and 51 captive Morelet's crocodiles (Crocodylus moreletii) in Campeche, Mexico, March-September 2007. Blood samples from 92 crocodiles (45 free-ranging and 47 captive) were collected for hematologic and plasma biochemical analyses. Average values of erythrocytes of free-ranging crocodiles were 1,046,166 cells/μl, and total white cells were 1.03 × 10(4) cells/μl. Captive crocodiles had erythrocyte and leukocyte values of 1,100,416 cells/μl and 8.51 × 10(3) cells/μl, respectively. There were no significant differences in values of erythrocytes or in hematocrit between free-ranging and captive crocodiles, or between sexes, or among size classes. Counts of leukocytes in free-ranging crocodiles were significantly higher than in captive individuals. The mean values of plasma analytes were 69.55 mg/l (glucose), 250.14 mg/l (cholesterol), 3.04 mg/l (uric acid), 2.70 mg/l (creatinine), and 20.20 IU/l (alanine aminotransferase). There were significant differences in cholesterol between free-ranging and captive crocodiles and between sexes.

  6. Pressurized Pepsin Digestion in Proteomics: An Automatable Alternative to Trypsin for Integrated Top-down Bottom-up Proteomics

    SciTech Connect

    Lopez-Ferrer, Daniel; Petritis, Konstantinos; Robinson, Errol W.; Hixson, Kim K.; Tian, Zhixin; Lee, Jung Hwa; Lee, Sang-Won; Tolic, Nikola; Weitz, Karl K.; Belov, Mikhail E.; Smith, Richard D.; Pasa-Tolic, Ljiljana

    2011-02-01

    Integrated top-down bottom-up proteomics combined with online digestion has great potential to improve the characterization of protein isoforms in biological systems and is amendable to highthroughput proteomics experiments. Bottom-up proteomics ultimately provides the peptide sequences derived from the tandem MS analyses of peptides after the proteome has been digested. Top-down proteomics conversely entails the MS analyses of intact proteins for more effective characterization of genetic variations and/or post-translational modifications (PTMs). Herein, we describe recent efforts towards efficient integration of bottom-up and top-down LCMS based proteomic strategies. Since most proteomic platforms (i.e. LC systems) operate in acidic environments, we exploited the compatibility of the pepsin (i.e. the enzyme’s natural acidic activity) for the integration of bottom-up and top-down proteomics. Pressure enhanced pepsin digestions were successfully performed and characterized with several standard proteins in either an offline mode using a Barocycler or an online mode using a modified high pressure LC system referred to as a fast online digestion system (FOLDS). FOLDS was tested using pepsin and a whole microbial proteome, and the results compared against traditional trypsin digestions on the same platform. Additionally, FOLDS was integrated with a RePlay configuration to demonstrate an ultra-rapid integrated bottom-up top-down proteomic strategy employing a standard mixture of proteins and a monkey pox virus proteome.

  7. A comparative analysis of human plasma and serum proteins by combining native PAGE, whole-gel slicing and quantitative LC-MS/MS: Utilizing native MS-electropherograms in proteomic analysis for discovering structure and interaction-correlated differences.

    PubMed

    Wen, Meiling; Jin, Ya; Manabe, Takashi; Chen, Shumin; Tan, Wen

    2017-09-04

    MS identification has long been used for PAGE-separated protein bands, but global and systematic quantitation utilizing MS after PAGE has remained rare and not been reported for native PAGE. Here we reported on a new method combining native PAGE, whole-gel slicing and quantitative LC-MS/MS, aiming at comparative analysis on not only abundance, but also structures and interactions of proteins. A pair of human plasma and serum samples were used as test samples and separated on a native PAGE gel. Six lanes of each sample were cut, each lane was further sliced into thirty-five 1.1 mm × 1.1 mm squares and all the squares were subjected to standardized procedures of in-gel digestion and quantitative LC-MS/MS. The results comprised 958 data rows that each contained abundance values of a protein detected in one square in eleven gel lanes (one plasma lane excluded). The data were evaluated to have satisfactory reproducibility of assignment and quantitation. Totally 315 proteins were assigned, with each protein assigned in 1-28 squares. The abundance distributions in the plasma and serum gel lanes were reconstructed for each protein, named as "native MS-electropherograms". Comparison of the electropherograms revealed significant plasma-versus-serum differences on 33 proteins in 87 squares (fold difference > 2 or < 0.5, p < 0.05). Many of the differences matched with accumulated knowledge on protein interactions and proteolysis involved in blood coagulation, complement and wound healing processes. We expect this method would be useful to provide more comprehensive information in comparative proteomic analysis, on both quantities and structures/interactions. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Metallurgical and chemical characterization of copper alloy reference materials within laser ablation inductively coupled plasma mass spectrometry: Method development for minimally-invasive analysis of ancient bronze objects

    NASA Astrophysics Data System (ADS)

    Walaszek, Damian; Senn, Marianne; Faller, Markus; Philippe, Laetitia; Wagner, Barbara; Bulska, Ewa; Ulrich, Andrea

    2013-01-01

    The chemical composition of ancient metal objects provides important information for manufacturing studies and authenticity verification of ancient copper or bronze artifacts. Non- or minimal-destructive analytical methods are preferred to mitigate visible damage. Laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) enables the determination of major elements as well as impurities down to lower ppm-levels, however, accuracy and precision of analysis strongly depend on the homogeneity of reference materials used for calibration. Moreover, appropriate analytical procedures are required e.g. in terms of ablation strategies (scan mode, spot size, etc.). This study reviews available copper alloy (certified) reference materials — (C)RMs from different sources and contributes new metallurgical data on homogeneity and spatial elemental distribution. Investigations of the standards were performed by optical and scanning electron microscopy with X-ray spectrometry (SEM-EDX) for the following copper alloy and bronze (certified) reference materials: NIST 454, BAM 374, BAM 211, BAM 227, BAM 374, BAM 378, BAS 50.01-2, BAS 50.03-4, and BAS 50.04-4. Additionally, the influence of inhomogeneities on different ablation and calibration strategies is evaluated to define an optimum analytical strategy in terms of line scan versus single spot ablation, variation of spot size, selection of the most appropriate RMs or minimum number of calibration reference materials.

  9. Temporal variance in hematologic and plasma biochemical reference intervals for free-ranging eastern box turtles (Terrapene carolina carolina).

    PubMed

    Kimble, Steven J A; Williams, Rod N

    2012-07-01

    Eastern box turtle (Terrapene carolina carolina) populations are in decline, likely due to anthropogenic forces and disease, necessitating hematologic and biochemical data from healthy individuals for evaluation of wild populations. We repeatedly sampled 21 free-ranging eastern box turtles from May to September 2009 in the spring, summer, and fall to establish temporal hematologic and biochemical reference intervals. Packed cell volume, aspartate aminotransferase, and potassium levels declined significantly as the active season progressed. High levels of albumin, globulin, and calcium coincided with the presence of eggs in females. These reference intervals should provide baseline data for the clinical evaluation of wild box turtles presented for veterinary care or for studies of wild populations.

  10. Ultra-High-Efficiency Strong Cation Exchange LC/RPLC/MS/MS for High Dynamic Range Characterization of the Human Plasma Proteome

    SciTech Connect

    Shen, Yufeng; Jacobs, Jon M.; Camp, David G.; Fang, Ruihua; Moore, Ronald J.; Smith, Richard D.; Xiao, Wenzhong; Davis, Ronald W.; Tompkins, Ronald G.

    2004-02-15

    In this study, we report a comprehensive approach for ultrahigh-efficiency separations by liquid chromatography (LC)/tandem mass spectrometry (MS/MS) for broad protein characterization of human plasma. The power of this approach is demonstrated by the confident identification of 1062 human plasma proteins based upon identification of 2992 tryptic peptides using highly conservative SEQUEST search criteria from a non-depleted human plasma sample. The approach provides a dynamic range of {approx}9 orders of magnitude in protein abundance using conventional ion trap MS/MS, which enabled identification of pg/mL concentration human plasma proteins (e.g. cytokines) co-existing with mg/mL-level human serum albumin. This dynamic range was obtained by combining high-efficiency reversed-phase (RP) LC coupled with efficient pre-fractionation strong cation exchange (SCX) LC to achieve ultrahigh-efficiency separations. A single-dimension, high-efficiency RPLC provided a protein identification dynamic range of 4 orders of magnitude in protein content and identified 433 human plasma proteins; while the ultrahigh-efficiency SCXLC/RPLC (i.e. 15 fractions from SCXLC), with the assistance of the SCXLC-sample component concentration (up to 102 fold), extended the protein identification dynamic range to {approx}9 orders of magnitude in protein content, identifying 822 human plasma proteins; combination of single- and two-dimension LC/MS/MS led to identification of 1062 human plasma proteins.

  11. Usefulness of Plasma Tissue Inhibitor of Matrix Metalloproteinase-4 to Predict Death and Myocardial Infarction in Patients With Diabetes Mellitus Referred for Coronary Angiography.

    PubMed

    Cavusoglu, Erdal; Kassotis, John T; Marmur, Jonathan D; Banerji, Mary Ann; Yanamadala, Sunitha; Chopra, Vineet; Anwar, Ayesha; Eng, Calvin

    2017-07-01

    TIMP-4 is the newest member of a family of secreted proteins known as the tissue inhibitors of metalloproteases that selectively inhibit matrix metalloproteases. TIMP-4 is abundantly expressed in human cardiovascular structures and has been implicated in cardiovascular disease. Furthermore, it has also been shown to be a novel target of peroxisome proliferator-activated receptor gamma in rat smooth muscle cells, suggesting a potential role in diabetes mellitus as well. However, there have been no studies that have specifically examined the utility of baseline plasma TIMP-4 levels for the prediction of long-term adverse cardiovascular outcomes. In this study, baseline plasma TIMP-4 levels were measured in 162 male patients with diabetes mellitus who were referred for coronary angiography and followed prospectively for the development of all-cause mortality and enzymatically confirmed myocardial infarction (MI) out to 5 years. After adjustment for a variety of baseline clinical, angiographic and laboratory parameters, plasma TIMP-4 levels were an independent predictor of all-cause mortality (hazard ratio 1.60, 95% CI 1.13 to 2.26; p = 0.0082) and MI (hazard ratio 1.61, 95% CI 1.19 to 2.18; p = 0.0021) at 5 years. Furthermore, in additional multivariate models that adjusted for a variety of biomarkers with established prognostic efficacy, TIMP-4 remained an independent predictor of adverse outcomes. In conclusion, elevated levels of TIMP-4 are associated with an increased risk of long-term all-cause mortality and MI in patients with diabetes mellitus referred for coronary angiography. Moreover, this association is independent of a variety of clinical, angiographic, and laboratory variables, including biomarkers with established prognostic efficacy in the prediction of adverse cardiovascular outcomes. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Laboratory blood analysis in Strigiformes-Part II: plasma biochemistry reference intervals and agreement between the Abaxis Vetscan V2 and the Roche Cobas c501.

    PubMed

    Ammersbach, Mélanie; Beaufrère, Hugues; Gionet Rollick, Annick; Tully, Thomas

    2015-03-01

    Limited plasma biochemical information is available in Strigiformes. Only one study investigated the agreement between a point-of-care with a reference laboratory analyzer for biochemistry variables in birds. The objective was to report reference intervals (RI) for plasma biochemistry variables in Strigiformes, and to assess agreement between the Abaxis Vetscan V2 and Roche Cobas c501. A prospective study was designed to assess plasma biochemistry RI for concentration of calcium, phosphorus, total protein, albumin, globulin, glucose, bilirubin, uric acid, bile acids, sodium, potassium, and chloride, and activities of AST, GGT, CK, amylase, lipase, LDH, and GLDH. In addition, the agreement between the Vetscan and the Cobas in owl species was assessed. A total of 190 individuals were sampled belonging to 12 Strigiformes species including Barn Owls, Barred Owls, Great Horned Owls, Eurasian Eagle Owls, Spectacled Owls, Eastern Screech Owls, Long-Eared Owls, Short-Eared Owls, Great Gray Owls, Snowy Owls, Northern Saw-Whet Owls, and Northern Hawk-Owls. Order-, species-, and method-specific RI were determined on both analyzers. Although Vetscan data were not equivalent to the Cobas, 4 analytes (glucose, AST, CK, and total protein, with correction for bias) were within acceptable agreement, 3 analytes (uric acid, calcium, and phosphorus) were within close agreement, and the remaining analytes were in strong disagreement. Species-specific differences were observed notably for the concentration of glucose in Barn Owls and electrolytes in Northern Saw-Whet Owls. Overall, this study suggests that the Vetscan has acceptable clinical performance in Strigiformes for some analytes and highlights discrepancies for several analytes. © 2015 American Society for Veterinary Clinical Pathology.

  13. Human biomonitoring reference values derived for persistent organic pollutants in blood plasma from the Canadian Health Measures Survey 2007-2011.

    PubMed

    Haines, Douglas A; Khoury, Cheryl; Saravanabhavan, Gurusankar; Werry, Kate; Walker, Mike; Malowany, Morie

    2017-06-01

    Nationally representative human biomonitoring data on persistent organic pollutants (POPs) including organochlorine pesticides (OCs), polychlorinated biphenyls (PCBs) brominated flame retardants (BFRs) and perfluoroalkyl substances (PFASs) are available through the Canadian Health Measures Survey (CHMS). We have used a systematic approach building on the reference interval concept proposed by the International Federation of Clinical Chemistry and Laboratory Medicine and the International Union of Pure and Applied Chemistry to derive human biomonitoring reference values (RV95s) for selected POPs in blood plasma in the general Canadian population. Biomarkers were chosen based on specific selection criteria including their detection in most Canadians (>66% detection rate). Age and sex were evaluated as possible partitioning criteria and separate RV95s were derived for the sub-populations in cases where partitioning was deemed necessary. RV95s for OCs, PCBs, and BFRs were derived both on a whole weight of blood plasma and on a lipid weight adjusted basis whereas they were derived only on a whole weight basis for PFASs. RV95s ranged from 0.018μg/L (PCB 201) to 21μg/L (perfluorooctane sulfonate) and from 3.1μg/kg lipid (PCB 201) to 1400μg/kg lipid (p,p'-DDE). The 22 RV95s reported in this paper represent the first set of reference values for POPs in the Canadian general population against which individual and population human biomonitoring data may be compared. Crown Copyright © 2017. Published by Elsevier GmbH. All rights reserved.

  14. CD5 molecule-like and transthyretin as putative biomarkers of chronic myeloid leukemia - an insight from the proteomic analysis of human plasma

    PubMed Central

    Fatima, Iram; Sadaf, Saima; Musharraf, Syed Ghulam; Hashmi, Naghma; Akhtar, Muhammad Waheed

    2017-01-01

    Better and sensitive biomarkers are needed to help understand the mechanism of disease onset, progression, prognosis and monitoring of the therapeutic response. Aim of this study was to identify the candidate circulating markers of chronic-phase chronic myeloid leukemia (CP-CML) manifestations, having potential to develop into predictive- or monitoring-biomarkers. A proteomic approach, two-dimensional gel electrophoresis in conjunction with mass spectrometry (2DE-MS), was employed for this purpose. Based on the spot intensity measurements, six proteins were found to be consistently dysregulated in CP-CML subjects compared to the healthy controls [false discovery rate (FDR) threshold ≤0.05]. These were identified as α-1-antichymotrypsin, α-1-antitrypsin, CD5 molecule-like, stress-induced phosphoprotein 1, vitamin D binding protein isoform 1 and transthyretin by MS analysis [PMF score ≥79; data accessible via ProteomeXchange with identifier PXD002757]. Quantitative ELISA, used for validation of candidate proteins both in the pre-treated and nilotinib-treated CP-CML cases, demonstrate that CD5 molecule-like, transthyretin and alpha-1-antitrypsin may serve as useful predictive markers and aid in monitoring the response of TKI-based therapy (ANOVA p < 0.0001). Two of the circulating marker proteins, identified in this study, had not previously been associated with chronic- or acute-phase myeloid leukemia. Exploration of their probable association with CP-CML, in a larger study cohort, may add to our understanding of the disease mechanism besides developing clinically useful biomarkers in future. PMID:28117336

  15. Large external quality assessment survey on thrombin generation with CAT: further evidence for the usefulness of normalisation with an external reference plasma.

    PubMed

    Perrin, Julien; Depasse, François; Lecompte, Thomas

    2015-07-01

    Calibrated Automated Thrombography (CAT) has been widely used to assess in vitro thrombin generation as an informative intermediary phenotype of coagulation. Interlaboratory exercises have documented a worrisome poor reproducibility. There are some data on the normalisation with an appropriate external reference plasma (RP). This multicentre study of the French-speaking CAT Club aimed at providing further evidence for the usefulness of such a normalisation. Lyophilised aliquots of a RP along with 3 plasmas (P1=normal; P2=hypo-; P3=hypercoagulable) were sent to 34 laboratories (corresponding to 38 instruments). CAT was studied using 1 and 5 pM tissue factor and other dedicated reagents. Normalisation with the local RP in use in the laboratory could also be performed. Interlaboratory CVs were calculated for each plasma before and after normalisation. Regarding endogenous thrombin potential, a good discrimination between the 3 plasmas was achieved in all laboratories but there was no overlap after normalisation only. CVs were generally not reduced with the use of local RP but were generally improved with normalisation using the external RP, often becoming lower than 10%. Regarding P2 however, the benefit of normalisation was poor, and there were analytical difficulties as well, some laboratories being unable to get a useable signal. We confirm that normalisation of CAT results with a suitable external RP is useful in "real life" practice as it often permits an acceptable level of interlaboratory variability. In case of frank hypocoagulability, further improvements are required to get reliable, potentially clinically relevant results. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Sub-cellular proteomics of Medicago truncatula

    PubMed Central

    Lee, Jeonghoon; Lei, Zhentian; Watson, Bonnie S.; Sumner, Lloyd W.

    2013-01-01

    Medicago truncatula is a leading model species and substantial molecular, genetic, genomics, proteomics, and metabolomics resources have been developed for this species to facilitate the study of legume biology. Currently, over 60 proteomics studies of M. truncatula have been published. Many of these have focused upon the unique symbiosis formed between legumes and nitrogen fixing rhizobia bacteria, while others have focused on seed development and the specialized proteomes of distinct tissues/organs. These include the characterization of sub-cellular organelle proteomes such as nuclei and mitochondria, as well as proteins distributed in plasma or microsomal membranes from various tissues. The isolation of sub-cellular proteins typically requires a series of steps that are labor-intensive. Thus, efficient protocols for sub-cellular fractionation, purification, and enrichment are necessary for each cellular compartment. In addition, protein extraction, solubilization, separation, and digestion prior to mass spectral identification are important to enhance the detection of low abundance proteins and to increase the overall detectable proportion of the sub-cellular proteome. This review summarizes the sub-cellular proteomics studies in M. truncatula. PMID:23641248

  17. Seasonal hematology and plasma biochemistry reference range values of the yellow-marginated box turtle (Cuora flavomarginata).

    PubMed

    Yang, Pu-Yu; Yu, Pin-Huan; Wu, Sheng-Hai; Chie, Chau-Hwa

    2014-06-01

    The yellow-marginated box turtle (Cuora flavomarginata) is critically endangered and very few studies have been performed to aid the conservation effort. This study reports the first complete analysis of blood parameters for this species and will provide a reference for future conservation studies. Hematology and biochemistry reference range values were established from 52 (18 males and 35 females) clinically healthy yellow-marginated box turtles (C. flavomarginata). In order to investigate the gender and seasonal differences in these ranges, blood samples were collected from the jugular veins of the turtles in January, April, July, and October to represent the four seasons. To detect significant variation (P < 0.05), data were analyzed using repeated measurements of analysis of variance. Significant gender differences were observed in red blood cell mass parameters, absolute eosinophils, absolute monocytes, alkaline phosphatase, aspartate aminotransferase, blood urea nitrogen, calcium, uric acid, cholesterol, creatine kinase, glucose, phosphorus (P), total protein, and triglycerides. Marked seasonal variations were noted in red blood cell mass parameters, absolute monocytes, albumin, alkaline phosphatase, aspartate aminotransferase, blood urea nitrogen, uric acid, creatine kinase, creatinine, glucose, P, and triglycerides. Primary reasons for the differences between gender and season were associated with reproduction and temperature change. Cuora flavomarginata has lower alanine aminotransferase values compared with other chelonians.

  18. Plasma electrophoretic profiles and hemoglobin binding protein reference intervals in the eastern box turtle (Terrapene carolina carolina) and influences of age, sex, season, and location.

    PubMed

    Flower, Jennifer E; Byrd, John; Cray, Carolyn; Allender, Matthew C

    2014-12-01

    Evaluation of plasma electrophoretic profiles and acute phase protein concentrations may play a valuable role in health assessment of reptiles; however, little is known about reference intervals in free-ranging eastern box turtles (Terrapene carolina carolina). The purpose of this study was to establish reference intervals of protein electrophoretic profiles and hemoglobin binding protein ([HBP] as determined by a haptoglobin assay) in free-ranging eastern box turtles and to assess any possible correlations between varying age class (adults vs. juvenile), sex (male, female, or unknown), season (spring, summer, or fall), or location (Tennessee vs. Illinois). Blood samples were obtained from 324 eastern box turtles from 2010 to 2012 at three sites in Illinois and one site in Tennessee, USA. Significant differences were observed with total protein (sex, season, state, Illinois location), albumin (age class, season, state, Illinois location), α-1 globulins (sex, season, Illinois location), α-2 globulins (sex, season, state, Illinois location), β globulins (age class, sex, season, state, Illinois location), γ globulins (sex, season state, Illinois location), and hemoglobin binding protein (age class, sex, state, Illinois location). The use of electrophoretic profiles and acute phase proteins is a relatively new concept in reptilian medicine, and this study allowed for establishment of references intervals in the eastern box turtle and emphasized differences that occured based on age, sex, season, and location. Future research in this area can now build on these data to determine changes in population health over time or alterations due to specific environmental or disease threats.

  19. Multiplying probe for accurate power measurements on an RF driven atmospheric pressure plasma jet applied to the COST reference microplasma jet

    NASA Astrophysics Data System (ADS)

    Beijer, P. A. C.; Sobota, A.; van Veldhuizen, E. M.; Kroesen, G. M. W.

    2016-03-01

    In this paper a new multiplying probe for measuring the power dissipated in a miniature capacitively coupled, RF driven, atmospheric pressure plasma jet (μAPPJ—COST Reference Microplasma Jet—COST RMJ) is presented. The approach aims for substantially higher accuracy than provided by traditionally applied methods using bi-directional power meters or commercially available voltage and current probes in conjunction with digitizing oscilloscopes. The probe is placed on a miniature PCB and designed to minimize losses, influence of unknown elements, crosstalk and variations in temperature. The probe is designed to measure powers of the order of magnitude of 0.1-10 W. It is estimated that it measures power with less than 2% deviation from the real value in the tested power range. The design was applied to measure power dissipated in COST-RMJ running in helium with a small addition of oxygen.

  20. Bracketing method with certified reference materials for high precision and accuracy determination of trace cadmium in drinking water by Inductively Coupled Plasma - Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Ketrin, Rosi; Handayani, Eka Mardika; Komalasari, Isna

    2017-01-01

    Two significant parameters to evaluate the measurement results are known as precision and accuracy. Both are associated with indeterminate and determinate error, respectively, that normally happen in such spectrometric measurement method as Inductively Coupled Plasma - Mass Spectrometry (ICP-MS). These errors must be eliminated or suppressed to get high precision and accuracy of the method. Decreasing the errors thus increasing the precision and accuracy of the method. In this study, bracketing method using two-point standard calibration was proposed in order to suppress the indeterminate error caused by instrumental drift thus increasing the result precision, and applied for measuring cadmium in drinking water samples. Certified reference material of ERM CA011b-Hard drinking water UK-metals was used to know the determinate error or measurement bias. When bias is obtained, some corrections are needed to get the accurate measurement result. The result was compared to that by external calibration method.

  1. An LC-MS/MS based candidate reference method for the quantification of total gentamicin in human serum and plasma using NMR characterized calibrator material.

    PubMed

    Lucha, Stephanie; Taibon, Judith; Pongratz, Stephan; Geletneky, Christian; Huber, Erasmus; Wintterle-Roehm, Christine; Lang, Robert; Grimm, Stefanie H; Duelffer, Thomas; Tarasov, Kirill; Zander, Johannes; Vogeser, Michael; Kobold, Uwe

    2017-01-01

    Accurate measurement of gentamicin concentration in serum and plasma is required for therapeutic drug monitoring to ensure appropriate treatment of patients. In this work, we present a validated LC-MS/MS-based candidate reference measurement procedure for total gentamicin quantification to be used for standardization and harmonization of routine assays applied for therapeutic drug monitoring of this compound. Total gentamicin is the sum of the concentrations of five known congeners C1, C1a, C2, C2a and C2b. To our knowledge, there is so far no LC-MS method for quantification of total gentamicin in human serum described in literature. Sample preparation was based on sample dilution with an aqueous internal standard solution followed by protein precipitation. Stable derivatives of gentamicin-glycine congeners were prepared by chemical synthesis and used as internal standards. The primary calibration material used in this assay was characterized by NMR spectroscopy and the pattern of the gentamicin congeners was determined. The total gentamicin was reported as the sum of the congeners which were quantified individually by LC-MS/MS. The method allows the measurement of total gentamicin in human serum and plasma in the concentration range of 0.1 to 12.0μg/ml with an assay imprecision of ≤6% CV and an assay accuracy between 96% and 114%. LOD and LOQ for the total gentamicin were 0.04μg/ml and 0.13μg/ml, respectively. Comparative measurement of 128 native patient samples using this method implemented at two laboratory sites showed an excellent agreement. Validation results proved that this protocol describes a robust and reliable method which is suggested as reference measurement procedure for the standardization and harmonization of routine assays for the quantification of total gentamicin. Copyright © 2016. Published by Elsevier B.V.

  2. Toward the Standardization of Mitochondrial Proteomics: The Italian Mitochondrial Human Proteome Project Initiative.

    PubMed

    Alberio, Tiziana; Pieroni, Luisa; Ronci, Maurizio; Banfi, Cristina; Bongarzone, Italia; Bottoni, Patrizia; Brioschi, Maura; Caterino, Marianna; Chinello, Clizia; Cormio, Antonella; Cozzolino, Flora; Cunsolo, Vincenzo; Fontana, Simona; Garavaglia, Barbara; Giusti, Laura; Greco, Viviana; Lucacchini, Antonio; Maffioli, Elisa; Magni, Fulvio; Monteleone, Francesca; Monti, Maria; Monti, Valentina; Musicco, Clara; Petrosillo, Giuseppe; Porcelli, Vito; Saletti, Rosaria; Scatena, Roberto; Soggiu, Alessio; Tedeschi, Gabriella; Zilocchi, Mara; Roncada, Paola; Urbani, Andrea; Fasano, Mauro

    2017-09-13

    The Mitochondrial Human Proteome Project aims at understanding the function of the mitochondrial proteome and its crosstalk with the proteome of other organelles. Being able to choose a suitable and validated enrichment protocol of functional mitochondria, based on the specific needs of the downstream proteomics analysis, would greatly help the researchers in the field. Mitochondrial fractions from ten model cell lines were prepared using three enrichment protocols and analyzed on seven different LC-MS/MS platforms. All data were processed using neXtProt as reference database. The data are available for the Human Proteome Project purposes through the ProteomeXchange Consortium with the identifier PXD007053. The processed data sets were analyzed using a suite of R routines to perform a statistical analysis and to retrieve subcellular and submitochondrial localizations. Although the overall number of identified total and mitochondrial proteins was not significantly dependent on the enrichment protocol, specific line to line differences were observed. Moreover, the protein lists were mapped to a network representing the functional mitochondrial proteome, encompassing mitochondrial proteins and their first interactors. More than 80% of the identified proteins resulted in nodes of this network but with a different ability in coisolating mitochondria-associated structures for each enrichment protocol/cell line pair.

  3. Determination of Ca, Mg, Na, Cd, Cu, Fe, K, Li and Zn in acid mine and reference water samples by inductively coupled plasma atomic fluorescence spectrometry

    USGS Publications Warehouse

    Sanzolone, R.F.; Meier, A.L.

    1986-01-01

    An inductively coupled plasma atomic fluorescence spectrometric (ICP-AFS) method was used for the determination of nine elements in natural water. Reference and acid mine water samples were analysed by this method to demonstrate its usefulness for hydrogeochemical exploration. The elements were determined in two groups based on the compatibility of operating conditions and consideration of element abundance levels in natural water. Ca, Mg and Na were determined as a group using one set of instrumental conditions and a 1 + 99 dilution of the sample, and Cd, Cu, Fe, K, Li and Zn were determined using another set of conditions and the undiluted sample. The detection limits for the elements are as follows: Ca, 1.4; Mg, 1.7; Na, 2.0; Cd, 1.8; Cu, 6.2; Fe, 15.8; K, 3.5; Li, 0.3; and Zn, 1.2 ng m1-1. Each element has a linear range spanning about four orders of magnitude. The method has good precision and accuracy, as shown by statistics on replicate analyses and by the agreement between values obtained and those recommended for the reference water samples, and also those obtained by atomic absorption spectrometry for the acid mine water samples.

  4. Completed | Office of Cancer Clinical Proteomics Research

    Cancer.gov

    Prior to the current Clinical Proteomic Tumor Analysis Consortium (CPTAC), previously funded initiatives associated with clinical proteomics research included: Clinical Proteomic Tumor Analysis Consortium (CPTAC 2.0) Clinical Proteomic Technologies for Cancer Initiative (CPTC) Mouse Proteomic Technologies Initiative

  5. ISPIDER Central: an integrated database web-server for proteomics.

    PubMed

    Siepen, Jennifer A; Belhajjame, Khalid; Selley, Julian N; Embury, Suzanne M; Paton, Norman W; Goble, Carole A; Oliver, Stephen G; Stevens, Robert; Zamboulis, Lucas; Martin, Nigel; Poulovassillis, Alexandra; Jones, Philip; Côté, Richard; Hermjakob, Henning; Pentony, Melissa M; Jones, David T; Orengo, Christine A; Hubbard, Simon J

    2008-07-01

    Despite the growing volumes of proteomic data, integration of the underlying results remains problematic owing to differences in formats, data captured, protein accessions and services available from the individual repositories. To address this, we present the ISPIDER Central Proteomic Database search (http://www.ispider.manchester.ac.uk/cgi-bin/ProteomicSearch.pl), an integration service offering novel search capabilities over leading, mature, proteomic repositories including PRoteomics IDEntifications database (PRIDE), PepSeeker, PeptideAtlas and the Global Proteome Machine. It enables users to search for proteins and peptides that have been characterised in mass spectrometry-based proteomics experiments from different groups, stored in different databases, and view the collated results with specialist viewers/clients. In order to overcome limitations imposed by the great variability in protein accessions used by individual laboratories, the European Bioinformatics Institute's Protein Identifier Cross-Reference (PICR) service is used to resolve accessions from different sequence repositories. Custom-built clients allow users to view peptide/protein identifications in different contexts from multiple experiments and repositories, as well as integration with the Dasty2 client supporting any annotations available from Distributed Annotation System servers. Further information on the protein hits may also be added via external web services able to take a protein as input. This web server offers the first truly integrated access to proteomics repositories and provides a unique service to biologists interested in mass spectrometry-based proteomics.

  6. ISPIDER Central: an integrated database web-server for proteomics

    PubMed Central

    Siepen, Jennifer A.; Belhajjame, Khalid; Selley, Julian N.; Embury, Suzanne M.; Paton, Norman W.; Goble, Carole A.; Oliver, Stephen G.; Stevens, Robert; Zamboulis, Lucas; Martin, Nigel; Poulovassillis, Alexandra; Jones, Philip; Côté, Richard; Hermjakob, Henning; Pentony, Melissa M.; Jones, David T.; Orengo, Christine A.; Hubbard, Simon J.

    2008-01-01

    Despite the growing volumes of proteomic data, integration of the underlying results remains problematic owing to differences in formats, data captured, protein accessions and services available from the individual repositories. To address this, we present the ISPIDER Central Proteomic Database search (http://www.ispider.manchester.ac.uk/cgi-bin/ProteomicSearch.pl), an integration service offering novel search capabilities over leading, mature, proteomic repositories including PRoteomics IDEntifications database (PRIDE), PepSeeker, PeptideAtlas and the Global Proteome Machine. It enables users to search for proteins and peptides that have been characterised in mass spectrometry-based proteomics experiments from different groups, stored in different databases, and view the collated results with specialist viewers/clients. In order to overcome limitations imposed by the great variability in protein accessions used by individual laboratories, the European Bioinformatics Institute's Protein Identifier Cross-Reference (PICR) service is used to resolve accessions from different sequence repositories. Custom-built clients allow users to view peptide/protein identifications in different contexts from multiple experiments and repositories, as well as integration with the Dasty2 client supporting any annotations available from Distributed Annotation System servers. Further information on the protein hits may also be added via external web services able to take a protein as input. This web server offers the first truly integrated access to proteomics repositories and provides a unique service to biologists interested in mass spectrometry-based proteomics. PMID:18440977

  7. Reference intervals for orotic acid in urine, plasma and dried blood spot using hydrophilic interaction liquid chromatography-tandem mass spectrometry.

    PubMed

    D'Apolito, Oceania; Garofalo, Daniela; la Marca, Giancarlo; Dello Russo, Antonio; Corso, Gaetano

    2012-02-01

    Orotic acid (OA), a marker of hereditary orotic aciduria, is usually used for the differential diagnosis of some hyperammonemic inherited defects of urea cycle and of basic amino acid transporters. This study was aimed to establish age related reference intervals of OA in urine, and for the first time in plasma, and dried blood spot (DBS) from 229 apparently healthy subjects aged from three days to 40 years. The quantification of OA was performed by a previously implemented method, using a stable isotope dilution with 1,3-[(15)N(2)]-orotic acid and hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS). The method has proved to be sensitive and accurate for a quantitative analysis of OA also in DBS and plasma. According to previous studies, urinary OA levels (mmol/mol of creatinine) decrease significantly with age. The upper limits (as 99th %ile) were of 3.44 and 1.30 in groups aged from three days to 1 year (group 1) and from 1 year to 12 years (group 2), respectively; in teenagers (from 13 to 19 years; group 3) and adults (from 20 to 40 years; group 4) urinary levels became more stable and the upper limits were of 0.64 and 1.21, respectively. Furthermore, OA levels in DBS (μM) also resulted significantly higher in subjects of group 1 (upper limit of 0.89) than in subjects of groups 2, 3 and 4 (upper limits of 0.24, 0.21, and 0.29, respectively). OA levels in plasma (μM) were significantly lower in subjects of group 3 (upper limit of 0.30) than in subjects of groups 1, 2, and 4 (upper limits of 0.59, 0.48, and 0.77, respectively). This method was also employed for OA quantification in plasma and DBS of 17 newborns affected by urea cycle defects, resulting sensitive and specific enough to screen these disorders.

  8. Soybean seed proteome rebalancing

    PubMed Central

    Herman, Eliot M.

    2014-01-01

    The soybean seed’s protein content and composition are regulated by both genetics and physiology. Overt seed protein content is specified by the genotype’s genetic framework and is selectable as a breeding trait. Within the genotype-specified protein content phenotype soybeans have the capacity to rebalance protein composition to create differing proteomes. Soybeans possess a relatively standardized proteome, but mutation or targeted engineering can induce large-scale proteome rebalancing. Proteome rebalancing shows that the output traits of seed content and composition result from two major types of regulation: genotype and post-transcriptional control of the proteome composition. Understanding the underlying mechanisms that specifies the seed proteome can enable engineering new phenotypes for the production of a high-quality plant protein source for food, feed, and industrial proteins. PMID:25232359

  9. Proteomics in diagnosis of prostate cancer.

    PubMed

    Davalieva, K; Polenakovic, M

    2015-01-01

    Prostate cancer (PCa) is the second most frequently diagnosed malignancy in men worldwide. The introduction of prostate specific antigen (PSA) has greatly increased the number of men diagnosed with PCa but at the same time, as a result of the low specificity, led to overdiagnosis, resulting to unnecessary biopsies and high medical cost treatments. The primary goal in PCa research today is to find a biomarker or biomarker set for clear and effecttive diagnosis of PCa as well as for distinction between aggressive and indolent cancers. Different proteomic technologies such as 2-D PAGE, 2-D DIGE, MALDI MS profiling, shotgun proteomics with label-based (ICAT, iTRAQ) and label-free (SWATH) quantification, MudPIT, CE-MS have been applied to the study of PCa in the past 15 years. Various biological samples, including tumor tissue, serum, plasma, urine, seminal plasma, prostatic secretions and prostatic-derived exosomes were analyzed with the aim of identifying diagnostic and prognostic biomarkers and developing a deeper understanding of the disease at the molecular level. This review is focused on the overall analysis of expression proteomics studies in the PCa field investigating all types of human samples in the search for diagnostics biomarkers. Emphasis is given on proteomics platforms used in biomarker discovery and characterization, explored sources for PCa biomarkers, proposed candidate biomarkers by comparative proteomics studies and the possible future clinical application of those candidate biomarkers in PCa screening and diagnosis. In addition, we review the specificity of the putative markers and existing challenges in the proteomics research of PCa.

  10. Proteomic patterns associated with heterosis.

    PubMed

    Xing, Jiewen; Sun, Qixin; Ni, Zhongfu

    2016-08-01

    Heterosis is characterized by higher seed yields, plant biomass or other traits in heterozygotes or hybrids compared with their genetically divergent parents, which are often homozygous. Despite extensive investigation of heterosis and its wide application in crops such as maize, rice, wheat and sorghum, its molecular basis is still enigmatic. In the past century, some pioneers have proposed multigene models referring to the complementation of allelic and gene expression variation, which is likely to be an important contributor to heterosis. In addition, there are potential interactions of epigenetic variation involved in heterosis via novel mechanisms. At the level of gene expression, many recent studies have revealed that the heterosis phenomenon can be deciphered not only at the transcriptional level but also at the proteomic level. This review presents an update on the information supporting the involvement of proteomic patterns in heterosis and a possible future direction of the field. This article is part of a Special Issue entitled: Plant Proteomics--a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock.

  11. Yeast Proteome Analysis

    NASA Astrophysics Data System (ADS)

    Matros, Andrea; Mock, Hans-Peter

    Yeast organisms, and specifically Saccharomyces cerevisiae, have become model systems for many aspects in fundamental and applied research. Consistently, many papers have been published applying proteome techniques to study these organisms. The review will give an overview on the proteome research performed on yeast systems so far; however, due to the large number of publications, only selected reports can be cited neglecting many more interesting ones in the interest of space. The review will focus on research involving mass spectrom-etry as a basic proteome technique, although many more approaches are relevant for the functional characterization of proteins in the cell, e.g. the yeast two-hybrid system. We will provide an overview on yeasts as models in the context of pro-teome analysis, and explain the basic techniques currently applied in proteome approaches. The main part of the review will deal with a survey on the current status of proteomic studies in yeasts. In a first part of this chapter, we will deal with the currently available proteome maps of yeasts, and in the following part we will discuss studies dealing with fundamental aspects, but also mention proteome studies related to applied microbiology. Finally, we will envisage future perspectives of the proteome technology for studying yeasts, and draw major conclusion on the current status reached in this field of functional genomics.

  12. In-Depth Profiling of the Peripheral Blood Mononuclear Cells Proteome for Clinical Blood Proteomics

    PubMed Central

    Končarević, Saša; Lößner, Christopher; Pike, Ian; Zucht, Hans-Dieter

    2014-01-01

    Peripheral blood mononuclear cells (PBMCs) are an easy accessible cellular part of the blood organ and, along with platelets, represent the only site of active gene expression in blood. These cells undergo immunophenotypic changes in various diseases and represent a peripheral source of monitoring gene expression and posttranslational modifications relevant to many diseases. Little is known about the source of many blood proteins and we hypothesise that release from PBMCs through active and passive mechanisms may account for a substantial part of the plasma proteome. The use of state-of-the-art proteomic profiling methods in PBMCs will enable minimally invasive monitoring of disease progression or response to treatment and discovery of biomarkers. To achieve this goal, detailed mapping of the PBMC proteome using a sensitive, robust, and quantitative methodological setup is required. We have applied an indepth gel-free proteomics approach using tandem mass tags (TMT), unfractionated and SCX fractionated PBMC samples, and LC-MS/MS with various modulations. This study represents a benchmark in deciphering the PBMC proteome as we provide a deep insight by identifying 4129 proteins and 25503 peptides. The identified proteome defines the scope that enables PBMCs to be characterised as cellular major biomarker pool within the blood organ. PMID:24724028

  13. In-depth profiling of the peripheral blood mononuclear cells proteome for clinical blood proteomics.

    PubMed

    Končarević, Saša; Lößner, Christopher; Kuhn, Karsten; Prinz, Thorsten; Pike, Ian; Zucht, Hans-Dieter

    2014-01-01

    Peripheral blood mononuclear cells (PBMCs) are an easy accessible cellular part of the blood organ and, along with platelets, represent the only site of active gene expression in blood. These cells undergo immunophenotypic changes in various diseases and represent a peripheral source of monitoring gene expression and posttranslational modifications relevant to many diseases. Little is known about the source of many blood proteins and we hypothesise that release from PBMCs through active and passive mechanisms may account for a substantial part of the plasma proteome. The use of state-of-the-art proteomic profiling methods in PBMCs will enable minimally invasive monitoring of disease progression or response to treatment and discovery of biomarkers. To achieve this goal, detailed mapping of the PBMC proteome using a sensitive, robust, and quantitative methodological setup is required. We have applied an indepth gel-free proteomics approach using tandem mass tags (TMT), unfractionated and SCX fractionated PBMC samples, and LC-MS/MS with various modulations. This study represents a benchmark in deciphering the PBMC proteome as we provide a deep insight by identifying 4129 proteins and 25503 peptides. The identified proteome defines the scope that enables PBMCs to be characterised as cellular major biomarker pool within the blood organ.

  14. Cell wall proteins: a new insight through proteomics.

    PubMed

    Jamet, Elisabeth; Canut, Hervé; Boudart, Georges; Pont-Lezica, Rafael F

    2006-01-01

    Cell wall proteins are essential constituents of plant cell walls; they are involved in modifications of cell wall components, wall structure, signaling and interactions with plasma membrane proteins at the cell surface. The application of proteomic approaches to the cell wall compartment raises important questions: are there technical problems specific to cell wall proteomics? What kinds of proteins can be found in Arabidopsis walls? Are some of them unexpected? What sort of post-translational modifications have been characterized in cell wall proteins to date? The purpose of this review is to discuss the experimental results obtained to date using proteomics, as well as some of the new questions challenging future research.

  15. CPTAC Biospecimen Collection Solicitation | Office of Cancer Clinical Proteomics Research

    Cancer.gov

    A funding opportunity in support of the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (CPTAC) seeks to prospectively procure tumor samples, collected for proteomics investigation. The scope of work under this Statement of Work encompasses the activities needed to prospectively procure high quality, clinically annotated human tumor samples, blood and plasma, and when feasible, normal tissue from volunteer patients suffering from colon, ovarian, and breast cancer.

  16. Collaborative study for establishment of a European Pharmacopoei Biological Reference Preparation (BRP) for B19 virus DNA testing of plasma pools by nucleic acid amplification technique.

    PubMed

    Nübling, C M; Daas, A; Buchheit, K H

    2004-01-01

    The goal of the collaborative study was to calibrate the B19 DNA content of a candidate Biological Reference Preparation (BRP) that is intended to be used for the validation of the analytical procedure, as threshold control and/or as quantitative reference material in the Nucleic Acid Amplification Technique (NAT) test of plasma pools for detection of B19 contamination. The candidate BRP was calibrated against the 1st International Standard for B19 DNA NAT assays. According to the European Pharmacopoeia monograph Human anti-D immunoglobulin, the threshold control needs to have a titre of 10( 4) IU/ml of B19 virus DNA. The lyophilised candidate BRP was prepared from 0.5 ml aliquots of a plasma pool spiked with B19 virus. The B19 virus originated from a "B19 virus window phase" blood donation (anti-B19 negative, B19-DNA high titre positive) and was diluted in a plasma pool tested negative by both serological and NAT assays for Hepatitis B Virus, Hepatitis C Virus and Human Immunodeficiency Virus 1 to obtain a B19-DNA concentration level in the range of 10( 6) copies/ml. The residual water content of the lyophilised candidate BRP was determined as 0.98 +/- 0.65% (mean +/- relative standard deviation). Sixteen laboratories (Official Medicine Control Laboratories, manufacturers of plasma derivatives, NAT test laboratories and NAT kit manufacturers) from nine countries participated. Participants were requested to test the candidate BRP and the International Standard (99/800) in four independent test runs on different days using their in-house qualitative and/or quantitative NAT methods. Sixteen laboratories reported results. Thirteen laboratories reported results from qualitative assays and 5 laboratories reported results from quantitative assays. Two laboratories reported results from both types of assay. For the qualitative assays a weighted combined potency of 5.64 log( 10) IU/ml with 95 per cent confidence limits of +/- 0.17 log( 10) which corresponds to 67 to 150

  17. Proteome of human plasma very low-density lipoprotein and low-density lipoprotein exhibits a link with coagulation and lipid metabolism.

    PubMed

    Dashty, M; Motazacker, M M; Levels, J; de Vries, M; Mahmoudi, M; Peppelenbosch, M P; Rezaee, F

    2014-03-03

    Apart from transporting lipids through the body, the human plasma lipoproteins very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) are also thought to serve as a modality for intra-organismal protein transfer, shipping proteins with important roles in inflammation and thrombosis from the site of synthesis to effector locations. To better understand the role of VLDL and LDL in the transport of proteins, we applied a combination of LTQ ORBITRAP-XL (nLC-MS/MS) with both in-SDS-PAGE gel and in-solution tryptic digestion of pure and defined VLDL and LDL fractions. We identified the presence of 95 VLDL- and 51 LDL-associated proteins including all known apolipoproteins and lipid transport proteins, and intriguingly a set of coagulation proteins, complement system and anti- microbial proteins. Prothrombin, protein S, fibrinogen γ, PLTP, CETP, CD14 and LBP were present on VLDL but not on LDL. Prenylcysteine oxidase 1, dermcidin, cathelicidin antimicrobial peptide, TFPI-1 and fibrinogen α chain were associated with both VLDL and LDL. Apo A-V is only present on VLDL and not on LDL. Collectively, this study provides a wealth of knowledge on the protein constituents of the human plasma lipoprotein system and strongly supports the notion that protein shuttling through this system is involved in the regulation of biological processes. Human diseases related to proteins carried by VLDL and LDL can be divided in three major categories: 1 - dyslipidaemia, 2 - atherosclerosis and vascular disease, and 3 - coagulation disorders.

  18. Proteomic plasma membrane profiling reveals an essential role for gp96 in the cell surface expression of LDLR family members, including the LDL receptor and LRP6.

    PubMed

    Weekes, Michael P; Antrobus, Robin; Talbot, Suzanne; Hör, Simon; Simecek, Nikol; Smith, Duncan L; Bloor, Stuart; Randow, Felix; Lehner, Paul J

    2012-03-02

    The endoplasmic reticulum chaperone gp96 is required for the cell surface expression of a narrow range of proteins, including toll-like receptors (TLRs) and integrins. To identify a more comprehensive repertoire of proteins whose cell surface expression is dependent on gp96, we developed plasma membrane profiling (PMP), a technique that combines SILAC labeling with selective cell surface aminooxy-biotinylation. This approach allowed us to compare the relative abundance of plasma membrane (PM) proteins on gp96-deficient versus gp96-reconstituted murine pre-B cells. Analysis of unfractionated tryptic peptides initially identified 113 PM proteins, which extended to 706 PM proteins using peptide prefractionation. We confirmed a requirement for gp96 in the cell surface expression of certain TLRs and integrins and found a marked decrease in cell surface expression of four members of the extended LDL receptor family (LDLR, LRP6, Sorl1 and LRP8) in the absence of gp96. Other novel gp96 client proteins included CD180/Ly86, important in the B-cell response to lipopolysaccharide. We highlight common structural motifs in these client proteins that may be recognized by gp96, including the beta-propeller and leucine-rich repeat. This study therefore identifies the extended LDL receptor family as an important new family of proteins whose cell surface expression is regulated by gp96.

  19. Proteomic Plasma Membrane Profiling Reveals an Essential Role for gp96 in the Cell Surface Expression of LDLR Family Members, Including the LDL Receptor and LRP6

    PubMed Central

    2012-01-01

    The endoplasmic reticulum chaperone gp96 is required for the cell surface expression of a narrow range of proteins, including toll-like receptors (TLRs) and integrins. To identify a more comprehensive repertoire of proteins whose cell surface expression is dependent on gp96, we developed plasma membrane profiling (PMP), a technique that combines SILAC labeling with selective cell surface aminooxy-biotinylation. This approach allowed us to compare the relative abundance of plasma membrane (PM) proteins on gp96-deficient versus gp96-reconstituted murine pre-B cells. Analysis of unfractionated tryptic peptides initially identified 113 PM proteins, which extended to 706 PM proteins using peptide prefractionation. We confirmed a requirement for gp96 in the cell surface expression of certain TLRs and integrins and found a marked decrease in cell surface expression of four members of the extended LDL receptor family (LDLR, LRP6, Sorl1 and LRP8) in the absence of gp96. Other novel gp96 client proteins included CD180/Ly86, important in the B-cell response to lipopolysaccharide. We highlight common structural motifs in these client proteins that may be recognized by gp96, including the beta-propeller and leucine-rich repeat. This study therefore identifies the extended LDL receptor family as an important new family of proteins whose cell surface expression is regulated by gp96. PMID:22292497

  20. Human liver proteome project: plan, progress, and perspectives.

    PubMed

    He, Fuchu

    2005-12-01

    The Human Liver Proteome Project is the first initiative of the human proteome project for human organs/tissues and aims at writing a modern Prometheus myth. Its global scientific objectives are to reveal the "solar system" of the human liver proteome, expression profiles, modification profiles, a protein linkage (protein-protein interaction) map, and a proteome localization map, and to define an ORFeome, physiome, and pathome. Since it was first proposed in April 2002, the Human Liver Proteome Project has attracted more than 100 laboratories from all over the world. In the ensuing 3 years, we set up a management infrastructure, identified reference laboratories, confirmed standard operating procedures, initiated international research collaborations, and finally achieved the first set of expression profile data.

  1. Proteome Profiling—Pitfalls and Progress

    PubMed Central

    Yates III, John R.

    2000-01-01

    In this review we examine the current state of analytical methods in proteomics. The conventional methodology using two-dimensional electrophoresis gels and mass spectrometry is discussed, with particular reference to the advantages and shortcomings thereof. Two recently published methods which offer an alternative approach are presented and discussed, with emphasis on how they can provide information not available via two-dimensional gel electrophoresis. These two methods are the isotope-coded affinity tags approach of Gygi et al. and the two-dimensional liquid chromatography–tandem mass spectrometry approach as presented by Link et al. We conclude that both of these new techniques represent significant advances in analytical methodology for proteome analysis. Furthermore, we believe that in the future biological research will continue to be enhanced by the continuation of such developments in proteomic analytical technology. PMID:10900454

  2. Advanced proteomic liquid chromatography

    SciTech Connect

    Xie, Fang; Smith, Richard D.; Shen, Yufeng

    2012-10-26

    Liquid chromatography coupled with mass spectrometry is the predominant platform used to analyze proteomics samples consisting of large numbers of proteins and their proteolytic products (e.g., truncated polypeptides) and spanning a wide range of relative concentrations. This review provides an overview of advanced capillary liquid chromatography techniques and methodologies that greatly improve separation resolving power and proteomics analysis coverage, sensitivity, and throughput.

  3. Quantitative plant proteomics.

    PubMed

    Bindschedler, Laurence V; Cramer, Rainer

    2011-02-01

    Quantitation is an inherent requirement in comparative proteomics and there is no exception to this for plant proteomics. Quantitative proteomics has high demands on the experimental workflow, requiring a thorough design and often a complex multi-step structure. It has to include sufficient numbers of biological and technical replicates and methods that are able to facilitate a quantitative signal read-out. Quantitative plant proteomics in particular poses many additional challenges but because of the nature of plants it also offers some potential advantages. In general, analysis of plants has been less prominent in proteomics. Low protein concentration, difficulties in protein extraction, genome multiploidy, high Rubisco abundance in green tissue, and an absence of well-annotated and completed genome sequences are some of the main challenges in plant proteomics. However, the latter is now changing with several genomes emerging for model plants and crops such as potato, tomato, soybean, rice, maize and barley. This review discusses the current status in quantitative plant proteomics (MS-based and non-MS-based) and its challenges and potentials. Both relative and absolute quantitation methods in plant proteomics from DIGE to MS-based analysis after isotope labeling and label-free quantitation are described and illustrated by published studies. In particular, we describe plant-specific quantitative methods such as metabolic labeling methods that can take full advantage of plant metabolism and culture practices, and discuss other potential advantages and challenges that may arise from the unique properties of plants.

  4. Enabling proteomic studies with RNA-Seq: The proteome of tomato pollen as a test case.

    PubMed

    Lopez-Casado, Gloria; Covey, Paul A; Bedinger, Patricia A; Mueller, Lukas A; Thannhauser, Theodore W; Zhang, Sheng; Fei, Zhangjun; Giovannoni, James J; Rose, Jocelyn K C

    2012-03-01

    Effective proteome profiling is generally considered to depend heavily on the availability of a high-quality DNA reference database. As such, proteomics has long been taxonomically restricted, with limited inroads being made into the proteomes of "non-model" organisms. However, next generation sequencing (NGS), and particularly RNA-Seq, now allows deep coverage detection of expressed genes at low cost, which in turn potentially facilitates the matching of peptide mass spectra with cognate gene sequence. To test this, we performed a quantitative analysis of the proteomes of pollen from domesticated tomato (Solanum lycopersicum) and two wild relatives that exhibit differences in mating systems and in interspecific reproductive barriers. Using a custom tomato RNA-Seq database created through 454 pyrosequencing, more than 1200 proteins were identified, with subsets showing expression differences between genotypes or in the accumulation of the corresponding transcripts. Importantly, no major qualitative or quantitative differences were observed in the characterized proteomes when mass spectra were used to interrogate either a highly curated community database of tomato sequences generated through traditional sequencing technologies, or the RNA-Seq database. We conclude that RNA-Seq provides a cost-effective and robust platform for protein identification and will be increasingly valuable to the field of proteomics. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Determination of trace sulfur in biodiesel and diesel standard reference materials by isotope dilution sector field inductively coupled plasma mass spectrometry.

    PubMed

    Amais, Renata S; Long, Stephen E; Nóbrega, Joaquim A; Christopher, Steven J

    2014-01-02

    A method is described for quantification of sulfur at low concentrations on the order of mgkg(-1) in biodiesel and diesel fuels using isotope dilution and sector field inductively coupled plasma mass spectrometry (ID-SF-ICP-MS). Closed vessel microwave-assisted digestion was employed using a diluted nitric acid and hydrogen peroxide decomposition medium to reduce sample dilution volumes. Medium resolution mode was employed to eliminate isobaric interferences at (32)S and (34)S related to polyatomic phosphorus and oxygen species, and sulfur hydride species. The method outlined yielded respective limits of detection (LOD) and limits of quantification (LOQ) of 0.7 mg kg(-1) S and 2.5 mg kg(-1) S (in the sample). The LOD was constrained by instrument background counts at (32)S but was sufficient to facilitate value assignment of total S mass fraction in NIST SRM 2723b Sulfur in Diesel Fuel Oil at 9.06±0.13 mg kg(-1). No statistically significant difference at a 95% confidence level was observed between the measured and certified values for certified reference materials NIST SRM 2773 B100 Biodiesel (Animal-Based), CENAM DRM 272b and NIST SRM 2723a Sulfur in Diesel Fuel Oil, validating method accuracy.

  6. Development of isotope dilution cold vapor inductively coupled plasma mass spectrometry and its application to the certification of mercury in NIST standard reference materials.

    PubMed

    Christopher, S J; Long, S E; Rearick, M S; Fassett, J D

    2001-05-15

    An isotope dilution cold vapor inductively coupled plasma mass spectrometry (ID-CV-ICPMS) method featuring gaseous introduction of mercury via tin chloride reduction has been developed and applied to the quantification and certification of mercury in various NIST standard reference materials: SRM 966 Toxic Metals in Bovine Blood (30 ng x mL(-1)); SRM 1641d Mercury in Water (1.6 microg x mL(-1)); and SRM 1946 Lake Superior Fish Tissue (436 ng x g(-1)). Complementary mercury data were generated for SRMs and NIST quality control standards using cold vapor atomic absorption spectroscopy (CVAAS). Certification results for the determination of mercury in SRM 1641d using two independent methods (ID-CV-ICPMS and CVAAS) showed a degree of agreement of 0.3% between the methods. Gaseous introduction of mercury into the ICPMS resulted in a single isotope sensitivity of 2 x 10(6) counts x s(-1)/ng x g(-1) for 201Hg and significantly reduced the memory and washout effects traditionally encountered in solution nebulization ICPMS. Figures of merit for isotope ratio accuracy and precision were evaluated at dwell times of 10, 20, 40, 80, and 160 ms using SRM 3133 Mercury Spectrometric Solution. The optimum dwell time of 80 ms yielded a measured 201Hg/202Hg isotope ratio within 0.13% of the theoretical natural value and a measurement precision of 0.34%, on the basis of three replicate injections of SRM 3133.

  7. Simultaneous determination of macro and trace elements in biological reference materials by microwave induced plasma optical emission spectrometry with slurry sample introduction

    NASA Astrophysics Data System (ADS)

    Matusiewicz, Henryk; Golik, Bartosz

    2004-05-01

    A slurry sampling technique (SST) has been utilized for simultaneous multi-element analysis by microwave-induced plasma optical emission spectrometry (MIP-OES). Slurry samples from a spray chamber are fed directly into the microwave cavity-torch assembly (power 300 W) with no desolvation apparatus. The performance of SST-MIP-OES was demonstrated by the determination of macro (Na, K, Ca, Mg, P) and trace (Cd, Cu, Mn, Sr, Zn) elements in three biological certified reference materials using a V-groove, clog-free Babington-type nebulizer. Slurry concentrations up to 1% m/v (particles <20 μm), prepared in 10% HNO 3 (pH 1.2) containing 0.01% of Triton X-100, were used with calibration by the standard additions method. The method offers relatively good precision (R.S.D. ranged from 7 to 11%) with measured concentrations being in satisfactory agreement with certified values for NRCC TORT-1 (Lobster hepatopancreas), NRCC LUTS-1 (Lobster hepatopancreas) and IAEA-153 (Milk powder). The concentrations of Na, K, Ca, Mg, P and Cd, Cu, Mn, Sr, Zn were determined in the range 90-22 000 μg/g and 1-420 μg/g, respectively. The method could be useful as a routine procedure.

  8. Survey of inorganic arsenic in marine animals and marine certified reference materials by anion exchange high-performance liquid chromatography-inductively coupled plasma mass spectrometry.

    PubMed

    Sloth, Jens J; Larsen, Erik H; Julshamn, Kåre

    2005-07-27

    A method for the determination of inorganic arsenic in seafood samples using high-performance liquid chromatography-inductively coupled plasma mass spectrometry is described. The principle of the method relied on microwave-assisted alkaline dissolution of the sample, which at the same time oxidized arsenite [As(III)] to arsenate [As(V)], whereby inorganic arsenic could be determined as the single species As(V). Anion exchange chromatography using isocratic elution with aqueous ammonium carbonate as the mobile phase was used for the separation of As(V) from other coextracted organoarsenic compounds, including arsenobetaine. The stability of organoarsenic compounds during the sample pretreatment was investigated, and no degradation/conversion to inorganic arsenic was detected. The method was employed for the determination of inorganic arsenic in a variety of seafood samples including fish, crustaceans, bivalves, and marine mammals as well as a range of marine certified reference materials, and the results were compared to values published in the literature. For fish and marine mammals, the results were in most cases below the limit of detection. For other sample types, inorganic arsenic concentrations up to 0.060 mg kg(-)(1) were found. In all samples, the inorganic arsenic content constituted less than 1% of the total arsenic content.

  9. Maintaining standardization: an update of the HUPO Brain Proteome Project.

    PubMed

    Hamacher, Michael; Stephan, Christian; Eisenacher, Martin; Hardt, Tanja; Marcus, Katrin; Meyer, Helmut E

    2008-04-01

    The Human Proteome Organisation was launched in February 2001 as a result of the need of an international proteomic forum to improve the understanding of human diseases. The initiative dealing with the brain is the Human Proteome Organisation Brain Proteome Project chaired in Germany and Korea. In order to estimate the existing approaches in brain proteomics, as well as to establish a standardized data reprocessing pipeline, pilot studies were initiated including both mouse and human samples. Data had to be submitted to a Data Collection Center for central re-processing and are now publicly accessible at the PRIDE database serving as reference data for future analysis. It became clear that heterogeneity, for example, different analysis strategies and data formats, are a real challenge when comparing results and when working in a consortium. Therefore, standardization, the organisation of data management and the synergistic effects of a consortium of collaborators are of outstanding importance to any big proteome analysis. The following manuscript will highlight these activities and aims of the Human Proteome Organisation Brain Proteome Project, summarizing its historical timeline and its two pilot studies.

  10. Proteomics in evolutionary ecology.

    PubMed

    Baer, B; Millar, A H

    2016-03-01

    Evolutionary ecologists are traditionally gene-focused, as genes propagate phenotypic traits across generations and mutations and recombination in the DNA generate genetic diversity required for evolutionary processes. As a consequence, the inheritance of changed DNA provides a molecular explanation for the functional changes associated with natural selection. A direct focus on proteins on the other hand, the actual molecular agents responsible for the expression of a phenotypic trait, receives far less interest from ecologists and evolutionary biologists. This is partially due to the central dogma of molecular biology that appears to define proteins as the 'dead-end of molecular information flow' as well as technical limitations in identifying and studying proteins and their diversity in the field and in many of the more exotic genera often favored in ecological studies. Here we provide an overview of a newly forming field of research that we refer to as 'Evolutionary Proteomics'. We point out that the origins of cellular function are related to the properties of polypeptide and RNA and their interactions with the environment, rather than DNA descent, and that the critical role of horizontal gene transfer in evolution is more about coopting new proteins to impact cellular processes than it is about modifying gene function. Furthermore, post-transcriptional and post-translational processes generate a remarkable diversity of mature proteins from a single gene, and the properties of these mature proteins can also influence inheritance through genetic and perhaps epigenetic mechanisms. The influence of post-transcriptional diversification on evolutionary processes could provide a novel mechanistic underpinning for elements of rapid, directed evolutionary changes and adaptations as observed for a variety of evolutionary processes. Modern state-of the art technologies based on mass spectrometry are now available to identify and quantify peptides, proteins, protein

  11. Proteomics approaches in the quest of kidney disease biomarkers.

    PubMed

    Frantzi, M; Bitsika, V; Charonis, A; Vlahou, A

    2011-01-01

    Proteomics refers to a group of analytical techniques for high throughput protein analysis, providing evidence for protein expression levels, subcellular localization, post-translational modifications and molecular interactions. As such, proteomics has contributed largely to our knowledge regarding molecular mechanisms underlying health and disease and pinpointed potential disease biomarkers. The scope of this review is to briefly introduce the principles of major proteomics techniques employed in biological research, including novel quantitative and molecular imaging mass spectrometry-based platforms. A few examples from the application of these techniques in biomarker discovery for kidney diseases are also provided.

  12. Characterization of Lipid Rafts from Medicago truncatula Root Plasma Membranes: A Proteomic Study Reveals the Presence of a Raft-Associated Redox System1[W

    PubMed Central

    Lefebvre, Benoit; Furt, Fabienne; Hartmann, Marie-Andrée; Michaelson, Louise V.; Carde, Jean-Pierre; Sargueil-Boiron, Françoise; Rossignol, Michel; Napier, Johnathan A.; Cullimore, Julie; Bessoule, Jean-Jacques; Mongrand, Sébastien

    2007-01-01

    Several studies have provided new insights into the role of sphingolipid/sterol-rich domains so-called lipid rafts of the plasma membrane (PM) from mammalian cells, and more recently from leaves, cell cultures, and seedlings of higher plants. Here we show that lipid raft domains, defined as Triton X-100-insoluble membranes, can also be prepared from Medicago truncatula root PMs. These domains have been extensively characterized by ultrastructural studies as well as by analysis of their content in lipids and proteins. M. truncatula lipid domains are shown to be enriched in sphingolipids and Δ7-sterols, with spinasterol as the major compound, but also in steryl glycosides and acyl-steryl glycosides. A large number of proteins (i.e. 270) have been identified. Among them, receptor kinases and proteins related to signaling, cellular trafficking, and cell wall functioning were well represented whereas those involved in transport and metabolism were poorly represented. Evidence is also given for the presence of a complete PM redox system in the lipid rafts. PMID:17337521

  13. Shotgun MS proteomic analysis of bronchoalveolar lavage fluid in normal subjects

    PubMed Central

    Nguyen, Elizabeth V.; Gharib, Sina A.; Schnapp, Lynn M.; Goodlett, David R.

    2014-01-01

    We provide a review of proteomic techniques used to characterize the bronchoalveolar lavage fluid (BALF) proteome of normal healthy subjects. Bronchoalveolar lavage (BAL) is the most common technique for sampling the components of the alveolar space. The proteomic techniques used to study normal BALF include protein separation by 2D gel electrophoresis whereby proteins were identified by comparison to a reference gel as well as high pressure liquid chromatography (HPLC)-tandem mass spectrometry technique, also known as shotgun proteomics. We summarize recent progress using shotgun MS technologies to define the normal BALF proteome. Surprisingly, we find that despite advances in shotgun proteomic technologies over the course of the last ten years, which have resulted in greater numbers of proteins being identified, the functional landscape of normal BALF proteome was similarly described by all methods examined. PMID:24616423

  14. Human fallopian tube proteome shows high coverage of mesenchymal stem cells associated proteins

    PubMed Central

    Wang, Chenyuan; Liu, Yang; Chang, Cheng; Wu, Songfeng; Gao, Jie; Zhang, Yang; Chen, Yingjie; Zhong, Fan; Deng, Gaopi

    2016-01-01

    The object of this research was to report a draft proteome of human fallopian tube (hFT) comprises 5416 identified proteins, which could be considered as a physiological reference to complement Human Proteome Draft. The proteomic raw data and metadata were stored in an integrated proteome resources centre iProX (IPX00034300). This hFT proteome contains many hFT markers newly identified by mass spectrum. This hFT proteome comprises 660 high-, 3605 medium- and 1181 low-abundant proteins. Ribosome, cytoskeleton, vesicle and protein folding associated proteins showed obvious tendency to be higher abundance in hFT. The extraordinary high coverage of mesenchymal stem cells (MSCs)-associated proteins were identified in this hFT proteome, which highly supported that hFT should contain a plenty of MSCs. PMID:26759384

  15. The proteome of sickle cell disease: insights from exploratory proteomic profiling

    PubMed Central

    Yuditskaya, Susan; Suffredini, Anthony F; J Kato, Gregory

    2011-01-01

    The expanding realm of exploratory proteomics has added a unique dimension to the study of the complex pathophysiology involved in sickle cell disease. A review of proteomic studies published on sickle cell erythrocytes and plasma shows trends of upregulation of antioxidant proteins, an increase in cytoskeletal defects, an increase in protein repair and turnover components, a decrease in lipid raft proteins and apolipoprotein dysregulation. Many of these findings are consistent with the pathophysiology of sickle cell disease, including high oxidant burden, resulting in damage to cytoskeletal and other proteins, and erythrocyte rigidity. More unexpected findings, such as a decrease in lipid raft components and apolipoprotein dysregulation, offer previously unexplored targets for future investigation and potential therapeutic intervention. Exploratory proteomic profiling is a valuable source of hypothesis generation for the cellular and molecular pathophysiology of sickle cell disease. PMID:21142886

  16. The effect of chronic kidney disease on the urine proteome in the domestic cat (Felis catus).

    PubMed

    Ferlizza, E; Campos, A; Neagu, A; Cuoghi, A; Bellei, E; Monari, E; Dondi, F; Almeida, A M; Isani, G

    2015-04-01

    Chronic kidney disease (CKD) is a major cause of mortality in cats, but sensitive and specific biomarkers for early prediction and monitoring of CKD are currently lacking. The present study aimed to apply proteomic techniques to map the urine proteome of the healthy cat and compare it with the proteome of cats with CKD. Urine samples were collected by cystocentesis from 23 healthy young cats and 17 cats with CKD. One-dimensional sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) was conducted on 4-12% gels. Two-dimensional electrophoresis (2DE) was applied to pooled urine samples from healthy cats (n = 4) and cats with CKD (n = 4), respectively. Sixteen protein bands and 36 spots were cut, trypsin-digested and identified by mass spectrometry. 1D-SDS-PAGE yielded an overall view of the protein profile and the separation of 32 ± 6 protein bands in the urine of healthy cats, while CKD cats showed significantly fewer bands (P < 0.01). 2-DE was essential in fractionation of the complex urine proteome, producing a reference map that included 20 proteins. Cauxin was the most abundant protein in urine of healthy cats. Several protease inhibitors and transport proteins that derive from plasma were also identified, including alpha-2-macroglobulin, albumin, transferrin, haemopexin and haptoglobin. There was differential expression of 27 spots between healthy and CKD samples (P < 0.05) and 13 proteins were unambiguously identified. In particular, increased expression of retinol-binding protein, cystatin M and apolipoprotein-H associated with decreased expression of uromodulin and cauxin confirmed tubular damage in CKD cats suggesting that these proteins are candidate biomarkers.

  17. Human Spermatozoa Quantitative Proteomic Signature Classifies Normo- and Asthenozoospermia.

    PubMed

    Saraswat, Mayank; Joenväärä, Sakari; Jain, Tushar; Tomar, Anil Kumar; Sinha, Ashima; Singh, Sarman; Yadav, Savita; Renkonen, Risto

    2017-01-01

    Scarcely understood defects lead to asthenozoospermia, which results in poor fertility outcomes. Incomplete knowledge of these defects hinders the development of new therapies and reliance on interventional therapies, such as in vitro fertilization, increases. Sperm cells, being transcriptionally and translationally silent, necessitate the proteomic approach to study the sperm function. We have performed a differential proteomics analysis of human sperm and seminal plasma and identified and quantified 667 proteins in sperm and 429 proteins in seminal plasma data set, which were used for further analysis. Statistical and mathematical analysis combined with pathway analysis and self-organizing maps clustering and correlation was performed on the data set.It was found that sperm proteomic signature combined with statistical analysis as opposed to the seminal plasma proteomic signature can differentiate the normozoospermic versus the asthenozoospermic sperm samples. This is despite the results that some of the seminal plasma proteins have big fold changes among classes but they fall short of statistical significance. S-Plot of the sperm proteomic data set generated some high confidence targets, which might be implicated in sperm motility pathways. These proteins also had the area under the curve value of 0.9 or 1 in ROC curve analysis.Various pathways were either enriched in these proteomic data sets by pathway analysis or they were searched by their constituent proteins. Some of these pathways were axoneme activation and focal adhesion assembly, glycolysis, gluconeogenesis, cellular response to stress and nucleosome assembly among others. The mass spectrometric data is available via ProteomeXchange with identifier PXD004098.

  18. Proteomics for systems toxicology

    PubMed Central

    Titz, Bjoern; Elamin, Ashraf; Martin, Florian; Schneider, Thomas; Dijon, Sophie; Ivanov, Nikolai V.; Hoeng, Julia; Peitsch, Manuel C.

    2014-01-01

    Current toxicology studies frequently lack measurements at molecular resolution to enable a more mechanism-based and predictive toxicological assessment. Recently, a systems toxicology assessment framework has been proposed, which combines conventional toxicological assessment strategies with system-wide measurement methods and computational analysis approaches from the field of systems biology. Proteomic measurements are an integral component of this integrative strategy because protein alterations closely mirror biological effects, such as biological stress responses or global tissue alterations. Here, we provide an overview of the technical foundations and highlight select applications of proteomics for systems toxicology studies. With a focus on mass spectrometry-based proteomics, we summarize the experimental methods for quantitative proteomics and describe the computational approaches used to derive biological/mechanistic insights from these datasets. To illustrate how proteomics has been successfully employed to address mechanistic questions in toxicology, we summarized several case studies. Overall, we provide the technical and conceptual foundation for the integration of proteomic measurements in a more comprehensive systems toxicology assessment framework. We conclude that, owing to the critical importance of protein-level measurements and recent technological advances, proteomics will be an integral part of integrative systems toxicology approaches in the future. PMID:25379146

  19. Proteomics in medical microbiology.

    PubMed

    Cash, P

    2000-04-01

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

  20. Proteomic Analysis of Plasma from California Sea Lions (Zalophus californianus) Reveals Apolipoprotein E as a Candidate Biomarker of Chronic Domoic Acid Toxicosis

    PubMed Central

    Neely, Benjamin A.; Ferrante, Jason A.; Chaves, J. Mauro; Soper, Jennifer L.; Almeida, Jonas S.; Arthur, John M.; Gulland, Frances M. D.; Janech, Michael G.

    2015-01-01

    Domoic acid toxicosis (DAT) in California sea lions (Zalophus californianus) is caused by exposure to the marine biotoxin domoic acid and has been linked to massive stranding events and mortality. Diagnosis is based on clinical signs in addition to the presence of domoic acid in body fluids. Chronic DAT further is characterized by reoccurring seizures progressing to status epilepticus. Diagnosis of chronic DAT is often slow and problematic, and minimally invasive tests for DAT have been the focus of numerous recent biomarker studies. The goal of this study was to retrospectively profile plasma proteins in a population of sea lions with chronic DAT and those without DAT using two dimensional gel electrophoresis to discover whether individual, multiple, or combinations of protein and clinical data could be utilized to identify sea lions with DAT. Using a training set of 32 sea lion sera, 20 proteins and their isoforms were identified that were significantly different between the two groups (p<0.05). Interestingly, 11 apolipoprotein E (ApoE) charge forms were decreased in DAT samples, indicating that ApoE charge form distributions may be important in the progression of DAT. In order to develop a classifier of chronic DAT, an independent blinded test set of 20 sea lions, seven with chronic DAT, was used to validate models utilizing ApoE charge forms and eosinophil counts. The resulting support vector machine had high sensitivity (85.7% with 92.3% negative predictive value) and high specificity (92.3% with 85.7% positive predictive value). These results suggest that ApoE and eosinophil counts along with machine learning can perform as a robust and accurate tool to diagnose chronic DAT. Although this analysis is specifically focused on blood biomarkers and routine clinical data, the results demonstrate promise for future studies combining additional variables in multidimensional space to create robust classifiers. PMID:25919366

  1. Proteomic Analysis of Plasma from California Sea Lions (Zalophus californianus) Reveals Apolipoprotein E as a Candidate Biomarker of Chronic Domoic Acid Toxicosis.

    PubMed

    Neely, Benjamin A; Ferrante, Jason A; Chaves, J Mauro; Soper, Jennifer L; Almeida, Jonas S; Arthur, John M; Gulland, Frances M D; Janech, Michael G

    2014-01-01

    Domoic acid toxicosis (DAT) in California sea lions (Zalophus californianus) is caused by exposure to the marine biotoxin domoic acid and has been linked to massive stranding events and mortality. Diagnosis is based on clinical signs in addition to the presence of domoic acid in body fluids. Chronic DAT further is characterized by reoccurring seizures progressing to status epilepticus. Diagnosis of chronic DAT is often slow and problematic, and minimally invasive tests for DAT have been the focus of numerous recent biomarker studies. The goal of this study was to retrospectively profile plasma proteins in a population of sea lions with chronic DAT and those without DAT using two dimensional gel electrophoresis to discover whether individual, multiple, or combinations of protein and clinical data could be utilized to identify sea lions with DAT. Using a training set of 32 sea lion sera, 20 proteins and their isoforms were identified that were significantly different between the two groups (p<0.05). Interestingly, 11 apolipoprotein E (ApoE) charge forms were decreased in DAT samples, indicating that ApoE charge form distributions may be important in the progression of DAT. In order to develop a classifier of chronic DAT, an independent blinded test set of 20 sea lions, seven with chronic DAT, was used to validate models utilizing ApoE charge forms and eosinophil counts. The resulting support vector machine had high sensitivity (85.7% with 92.3% negative predictive value) and high specificity (92.3% with 85.7% positive predictive value). These results suggest that ApoE and eosinophil counts along with machine learning can perform as a robust and accurate tool to diagnose chronic DAT. Although this analysis is specifically focused on blood biomarkers and routine clinical data, the results demonstrate promise for future studies combining additional variables in multidimensional space to create robust classifiers.

  2. Indirect reference intervals of plasma and serum thyrotropin (TSH) concentrations from intra-laboratory data bases from several German and Italian medical centres.

    PubMed

    Arzideh, Farhad; Wosniok, Werner; Haeckel, Rainer

    2011-04-01

    The dogma of establishing intra-laboratory reference limits (RLs) and their periodic review cannot be fulfilled by most laboratories due to the expenses involved. Thus, most laboratories adopt external sources for their RLs, often neglecting the problems of transferability. This is particularly problematic for analytes with a large diversity of existing RLs, as for example thyrotropin (TSH). Several attempts were taken to derive RLs from the large data pools stored in modern laboratory information systems. These attempts were further developed to a more sophisticated indirect procedure. The new approach can be considered a combined concept because it pre-excludes some subjects by direct criteria a-posterior. In the current study, the applicability of the new concept for modern protein bindings assays was examined for estimating RLs of serum and plasma TSH with data sets from several German and Italian laboratories. A smoothed kernel density function was estimated for the distribution of the total mixed data of the sample group (combined data of non-diseased and diseased subjects). It was assumed that the "central" part of the distribution of all data represents the non-diseased ("healthy") population. The central part was defined by truncation points using an optimisation method, and was used to estimate a Gaussian distribution of the values of presumably non-diseased subjects after Box-Cox transformation of the empirical data. This distribution was now considered as the distribution of the non-diseased subgroup. The percentiles of this parametrical distribution were calculated to obtain RLs. RLs determined by the indirect combined decomposition technique led to similar RLs as found by several recent study reports using a direct method according to international recommendations. Furthermore, the RLs obtained from 13 laboratories in two different European regions reflected the well-known differences of various analytical procedures. Stratification for gender and age

  3. Determination of trace elements by inductively coupled plasma mass spectrometry of biomass and fuel oil reference materials using milligram sample sizes.

    PubMed

    Lachas, H; Richaud, R; Herod, A A; Dugwell, D R; Kandiyoti, R

    2000-01-01

    Most of the analytical techniques used to quantify elements associated with solid samples suffer from high detection limits and cannot be used for trace elements in biomass samples, particularly when only 20 mg are available for analysis. Inductively coupled plasma mass spectrometry (ICP-MS) can achieve detection limits of parts-per-trillion with liquid sample introduction by solution nebulisation. This technique was therefore tested with two standard biomass reference materials: oriental tobacco leaves and cabbage leaves. Two preparations successfully used on coal standards were used to digest the solid samples: a total digestion method (wet ashing digestion) and a partial leaching (microwave extraction). The concentrations of up to seventeen elements (As, Ba, Be, Cd, Co, Cr, Cu, Ga, Mn, Mo, Ni, Pb, Sb, Se, Sn, V and Zn) were measured after the two preparations. The accuracy and sensitivity of the measurements improved when the dilution factor decreased from 5000 to 1000 and to 500. Since the proportion of mineral matter in biomass samples is small (5%), the microwave digestion extracted elements that are generally not completely extracted from coal samples (e.g. Sb). However, some trace element concentrations were below the limit of quantification after microwave extraction, even with a reduced dilution factor (As, Se and Mo) and could not be quantified. A fuel oil was also digested. The trace element concentrations were very low (between 28 and 0.1 microgram g(-1)) but acceptable results were obtained by applying a dilution factor of 100. Only six elements in the fuel oil (As, Ba, Co, Ni, Se and V) had certified or indicated values. Factors affecting the accuracy and sensitivity of the analyses are discussed. The reproducibility of analysis of the tobacco leaf standard was checked over a period of nine months by both digestion methods. The wet ashing method gave acceptable reproducibility for Ba, Cd, Co, Cu, Ga, Mn, Mo, Ni, Pb, V and Zn but poor precision for Cr

  4. Proteome sequencing goes deep

    PubMed Central

    Richards, Alicia L.; Merrill, Anna E.; Coon, Joshua J.

    2014-01-01

    Advances in mass spectrometry have transformed the scope and impact of protein characterization efforts. Identifying hundreds of proteins from rather simple biological matrices, such as yeast, was a daunting task just a few decades ago. Now, expression of more than half of the estimated ~20,000 human protein coding genes can be confirmed in record time and from minute sample quantities. Access to proteomic information at such unprecedented depths has been fueled by strides in every stage of the shotgun proteomics workflow – from sample processing to data analysis – and promises to revolutionize our understanding of the causes and consequences of proteome variation. PMID:25461719

  5. Proteomic Findings in Melanoma

    PubMed Central

    Sengupta, Deepanwita; Tackett, Alan J

    2016-01-01

    Although the emergence of proteomics as an independent branch of science is fairly recent, within a short period of time it has contributed substantially in various disciplines. The tool of mass spectrometry has become indispensable in the analysis of complex biological samples. Clinical applications of proteomics include detection of predictive and diagnostic markers, understanding mechanism of action of drugs as well as resistance mechanisms against them and assessment of therapeutic efficacy and toxicity of drugs in patients. Here, we have summarized the major contributions of proteomics towards the study of melanoma, which is a deadly variety of skin cancer with a high mortality rate. PMID:27274624

  6. Shotgun proteomics in neuroscience.

    PubMed

    Liao, Lujian; McClatchy, Daniel B; Yates, John R

    2009-07-16

    Mass spectrometry-based proteomics is increasingly used to address basic and clinical questions in biomedical research through studies of differential protein expression, protein-protein interactions, and posttranslational modifications. The complex structural and functional organization of the human brain warrants the application of high-throughput, systematic approaches to understand the functional alterations under normal physiological conditions and the perturbations of neurological diseases. This primer focuses on shotgun-proteomics-based tandem mass spectrometry for the identification of proteins in a complex mixture. It describes the basic concepts of protein differential expression analysis and posttranslational modification analysis and discusses several strategies to improve the coverage of the proteome.