Science.gov

Sample records for reference plasma proteome

  1. Blood plasma reference material: a global resource for proteomic research.

    PubMed

    Malm, Johan; Danmyr, Pia; Nilsson, Rolf; Appelqvist, Roger; Végvári, Akos; Marko-Varga, György

    2013-07-01

    There is an ever-increasing awareness and interest within the clinical research field, creating a large demand for blood fraction samples as well as other clinical samples. The translational research area is another field that is demanding for blood samples, used widely in proteomics, genomics, as well as metabolomics. Blood samples are globally the most common biological samples that are used in a broad variety of applications in life science. We hereby introduce a new reference blood plasma standard (heparin) that is aimed as a global resource for the proteomics community. We have developed these reference plasma standards by defining the Control group as those with C-reactive protein levels <3 mg/L and a Disease group with C-reactive protein ranges >30 mg/L. In these references we have used both newborn children 1-2 weeks, as well as youngsters 15-30 years, and middle aged 30-50 years, and elderly patients at the ages of 65+. In total, there were 80 patients in each group in the reference plasma pools. We provide data on the developments and characteristics of the reference blood plasma standards, as well as what is used by the team members at the respective laboratories. The standards have been evaluated by pilot sample processing in biobanking operations and are currently a resource that allows the Proteomic society to perform quantitative proteomic studies. By the use of high quality reference plasma samples, global initiatives, such as the Chromosome Human Proteome Project (C-HPP), will benefit as one scientific program when the entire human proteome is mapped and linked to human diseases. The plasma reference standards are a global resource and can be accessed upon request. PMID:23701512

  2. 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

    2010-09-01

    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.

  3. Depletion of Abundant Plasma Proteins and Limitations of Plasma Proteomics

    PubMed Central

    Tu, Chengjian; Rudnick, Paul A.; Martinez, Misti Y.; Cheek, Kristin L.; Stein, Stephen E.; Slebos, Robbert J. C.; Liebler, Daniel C.

    2010-01-01

    Immunoaffinity depletion with antibodies to the top 7 or top 14 high abundance plasma proteins is used to enhance detection of lower abundance proteins in both shotgun and targeted proteomic analyses. We evaluated the effects of top 7/top 14 immunodepletion on the shotgun proteomic analysis of human plasma. Our goal was to evaluate the impact of immunodepletion on detection of proteins across detectable ranges of abundance. The depletion columns afforded highly repeatable and efficient plasma protein fractionation. Relatively few nontargeted proteins were captured by the depletion columns. Analyses of unfractionated and immunodepleted plasma by peptide isoelectric focusing (IEF), followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) demonstrated enrichment of nontargeted plasma proteins by an average of 4-fold, as assessed by MS/MS spectral counting. Either top 7 or top 14 immunodepletion resulted in a 25% increase in identified proteins compared to unfractionated plasma. Although 23 low abundance (<10 ng mL−1) plasma proteins were detected, they accounted for only 5–6% of total protein identifications in immunodepleted plasma. In both unfractionated and immunodepleted plasma, the 50 most abundant plasma proteins accounted for 90% of cumulative spectral counts and precursor ion intensities, leaving little capacity to sample lower abundance proteins. Untargeted proteomic analyses using current LC-MS/MS platforms—even with immunodepletion—cannot be expected to efficiently discover low abundance, disease-specific biomarkers in plasma. PMID:20677825

  4. 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

  5. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  7. 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

  8. Additions to the Human Plasma Proteome via a Tandem MARS Depletion iTRAQ-Based Workflow.

    PubMed

    Cao, Zhiyun; Yende, Sachin; Kellum, John A; Robinson, Renã A S

    2013-01-01

    Robust platforms for determining differentially expressed proteins in biomarker and discovery studies using human plasma are of great interest. While increased depth in proteome coverage is desirable, it is associated with costs of experimental time due to necessary sample fractionation. We evaluated a robust quantitative proteomics workflow for its ability (1) to provide increased depth in plasma proteome coverage and (2) to give statistical insight useful for establishing differentially expressed plasma proteins. The workflow involves dual-stage immunodepletion on a multiple affinity removal system (MARS) column, iTRAQ tagging, offline strong-cation exchange chromatography, and liquid chromatography tandem mass spectrometry (LC-MS/MS). Independent workflow experiments were performed in triplicate on four plasma samples tagged with iTRAQ 4-plex reagents. After stringent criteria were applied to database searched results, 689 proteins with at least two spectral counts (SC) were identified. Depth in proteome coverage was assessed by comparison to the 2010 Human Plasma Proteome Reference Database in which our studies reveal 399 additional proteins which have not been previously reported. Additionally, we report on the technical variation of this quantitative workflow which ranges from ±11 to 30%.

  9. Additions to the Human Plasma Proteome via a Tandem MARS Depletion iTRAQ-Based Workflow

    PubMed Central

    Cao, Zhiyun; Yende, Sachin; Kellum, John A.; Robinson, Renã A. S.

    2013-01-01

    Robust platforms for determining differentially expressed proteins in biomarker and discovery studies using human plasma are of great interest. While increased depth in proteome coverage is desirable, it is associated with costs of experimental time due to necessary sample fractionation. We evaluated a robust quantitative proteomics workflow for its ability (1) to provide increased depth in plasma proteome coverage and (2) to give statistical insight useful for establishing differentially expressed plasma proteins. The workflow involves dual-stage immunodepletion on a multiple affinity removal system (MARS) column, iTRAQ tagging, offline strong-cation exchange chromatography, and liquid chromatography tandem mass spectrometry (LC-MS/MS). Independent workflow experiments were performed in triplicate on four plasma samples tagged with iTRAQ 4-plex reagents. After stringent criteria were applied to database searched results, 689 proteins with at least two spectral counts (SC) were identified. Depth in proteome coverage was assessed by comparison to the 2010 Human Plasma Proteome Reference Database in which our studies reveal 399 additional proteins which have not been previously reported. Additionally, we report on the technical variation of this quantitative workflow which ranges from ±11 to 30%. PMID:23509626

  10. 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.

  11. Proteome Dynamics Reveals Pro-Inflammatory Remodeling of Plasma Proteome in a Mouse Model of NAFLD.

    PubMed

    Li, Ling; Bebek, Gurkan; Previs, Stephen F; Smith, Jonathan D; Sadygov, Rovshan G; McCullough, Arthur J; Willard, Belinda; Kasumov, Takhar

    2016-09-01

    Nonalcoholic fatty liver disease (NAFLD) is associated with an increased risk of cardiovascular disease. Because the liver is the major source of circulatory proteins, it is not surprising that hepatic disease could lead to alterations in the plasma proteome, which are therein implicated in atherosclerosis. The current study used low-density lipoprotein receptor-deficient (LDLR(-/-)) mice to examine the impact of Western diet (WD)-induced NAFLD on plasma proteome homeostasis. Using a (2)H2O-metabolic labeling method, we found that a WD led to a proinflammatory distribution of circulatory proteins analyzed in apoB-depleted plasma, which was attributed to an increased production. The fractional turnover rates of short-lived proteins that are implicated in stress-response, lipid metabolism, and transport functions were significantly increased with WD (P < 0.05). Pathway analyses revealed that alterations in plasma proteome dynamics were related to the suppression of hepatic PPARα, which was confirmed based on reduced gene and protein expression of PPARα in mice fed a WD. These changes were associated with ∼4-fold increase (P < 0.0001) in the proinflammatory property of apoB-depleted plasma. In conclusion, the proteome dynamics method reveals proinflammatory remodeling of the plasma proteome relevant to liver disease. The approach used herein may provide a useful metric of in vivo liver function and better enable studies of novel therapies surrounding NAFLD and other diseases. PMID:27439437

  12. Extensive dataset of boar seminal plasma proteome displaying putative reproductive functions of identified proteins.

    PubMed

    Perez-Patiño, Cristina; Barranco, Isabel; Parrilla, Inmaculada; Martinez, Emilio A; Rodriguez-Martinez, Heriberto; Roca, Jordi

    2016-09-01

    A complete proteomic profile of seminal plasma (SP) remains challenging, particularly in porcine. The data reports on the analysis of boar SP-proteins by using a combination of SEC, 1-D SDS PAGE and NanoLC-ESI-MS/MS from 33 pooled SP-samples (11 boars, 3 ejaculates/boar). A complete dataset of the 536 SP-proteins identified and validated with confidence ≥95% (Unused Score >1.3) and a false discovery rate (FDR) ≤1%, is provided. In addition, the relative abundance of 432 of them is also shown. Gene ontology annotation of the complete SP-proteome complemented by an extensive description of the putative reproductive role of SP-proteins, providing a valuable source for a better understanding of SP role in the reproductive success. This data article refers to the article entitled "Characterization of the porcine seminal plasma proteome comparing ejaculate portions" (Perez-Patiño et al., 2016) [1]. PMID:27583342

  13. 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.

  14. 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.

  15. Statistical Analysis of Variation in the Human Plasma Proteome

    DOE PAGES

    Corzett, Todd H.; Fodor, Imola K.; Choi, Megan W.; Walsworth, Vicki L.; Turteltaub, Kenneth W.; McCutchen-Maloney, Sandra L.; Chromy, Brett A.

    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

  16. 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

  17. Large-scale and high-confidence proteomic analysis of human seminal plasma

    PubMed Central

    Pilch, Bartosz; Mann, Matthias

    2006-01-01

    Background The development of mass spectrometric (MS) techniques now allows the investigation of very complex protein mixtures ranging from subcellular structures to tissues. Body fluids are also popular targets of proteomic analysis because of their potential for biomarker discovery. Seminal plasma has not yet received much attention from the proteomics community but its characterization could provide a future reference for virtually all studies involving human sperm. The fluid is essential for the survival of spermatozoa and their successful journey through the female reproductive tract. Results Here we report the high-confidence identification of 923 proteins in seminal fluid from a single individual. Fourier transform MS enabled parts per million mass accuracy, and two consecutive stages of MS fragmentation allowed confident identification of proteins even by single peptides. Analysis with GoMiner annotated two-thirds of the seminal fluid proteome and revealed a large number of extracellular proteins including many proteases. Other proteins originated from male accessory glands and have important roles in spermatozoan survival. Conclusion This high-confidence characterization of seminal plasma content provides an inventory of proteins with potential roles in fertilization. When combined with quantitative proteomics methodologies, it should be useful for studies of fertilization, male infertility, and prostatic and testicular cancers. PMID:16709260

  18. Bovine sperm plasma membrane proteomics through biotinylation and subcellular enrichment.

    PubMed

    Kasvandik, Sergo; Sillaste, Gerly; Velthut-Meikas, Agne; Mikelsaar, Aavo-Valdur; Hallap, Triin; Padrik, Peeter; Tenson, Tanel; Jaakma, Ülle; Kõks, Sulev; Salumets, Andres

    2015-06-01

    A significant proportion of mammalian fertilization is mediated through the proteomic composition of the sperm surface. These protein constituents can present as biomarkers to control and regulate breeding of agricultural animals. Previous studies have addressed the bovine sperm cell apical plasma membrane (PM) proteome with nitrogen cavitation enrichment. Alternative workflows would enable to expand the compositional data more globally around the entire sperm's surface. We used a cell surface biotin-labeling in combination with differential centrifugation to enrich sperm surface proteins. Using nano-LC MS/MS, 338 proteins were confidently identified in the PM-enriched proteome. Functional categories of sperm-egg interaction, protein turnover, metabolism as well as molecular transport, spermatogenesis, and signal transduction were represented by proteins with high quantitative signal in our study. A highly significant degree of enrichment was found for transmembrane and PM-targeted proteins. Among them, we also report proteins previously not described on bovine sperm (CPQ, CD58, CKLF, CPVL, GLB1L3, and LPCAT2B) of which CPQ and CPVL cell surface localization was further validated. A descriptive overview of the bovine sperm PM integral and peripheral proteins is provided to complement future studies on animal reproduction and its relation to sperm cell surface. All MS data have been deposited in the ProteomeXchange with identifier PXD001096 (http://proteomecentral.proteomexchange.org/dataset/PXD001096).

  19. Quantitative proteomics analysis of adsorbed plasma proteins classifies nanoparticles with different surface properties and size

    SciTech Connect

    Zhang, Haizhen; Burnum, Kristin E.; Luna, Maria L.; Petritis, Brianne O.; Kim, Jong Seo; Qian, Weijun; Moore, Ronald J.; Heredia-Langner, Alejandro; Webb-Robertson, Bobbie-Jo M.; Thrall, Brian D.; Camp, David G.; Smith, Richard D.; Pounds, Joel G.; Liu, Tao

    2011-12-01

    In biofluids (e.g., blood plasma) nanoparticles are readily embedded in layers of proteins that can affect their biological activity and biocompatibility. Herein, we report a study on the interactions between human plasma proteins and nanoparticles with a controlled systematic variation of properties using stable isotope labeling and liquid chromatography-mass spectrometry (LC-MS) based quantitative proteomics. Novel protocol has been developed to simplify the isolation of nanoparticle bound proteins and improve the reproducibility. Plasma proteins associated with polystyrene nanoparticles with three different surface chemistries and two sizes as well as for four different exposure times (for a total of 24 different samples) were identified and quantified by LC-MS analysis. Quantitative comparison of relative protein abundances were achieved by spiking an 18 O-labeled 'universal reference' into each individually processed unlabeled sample as an internal standard, enabling simultaneous application of both label-free and isotopic labeling quantitation across the sample set. Clustering analysis of the quantitative proteomics data resulted in distinctive pattern that classifies the nanoparticles based on their surface properties and size. In addition, data on the temporal study indicated that the stable protein 'corona' that was isolated for the quantitative analysis appeared to be formed in less than 5 minutes. The comprehensive results obtained herein using quantitative proteomics have potential implications towards predicting nanoparticle biocompatibility.

  20. Cardiovascular-related proteins identified in human plasma by the HUPO Plasma Proteome Project pilot phase.

    PubMed

    Berhane, Beniam T; Zong, Chenggong; Liem, David A; Huang, Aaron; Le, Steven; Edmondson, Ricky D; Jones, Richard C; Qiao, Xin; Whitelegge, Julian P; Ping, Peipei; Vondriska, Thomas M

    2005-08-01

    Proteomic profiling of accessible bodily fluids, such as plasma, has the potential to accelerate biomarker/biosignature development for human diseases. The HUPO Plasma Proteome Project pilot phase examined human plasma with distinct proteomic approaches across multiple laboratories worldwide. Through this effort, we confidently identified 3020 proteins, each requiring a minimum of two high-scoring MS/MS spectra. A critical step subsequent to protein identification is functional annotation, in particular with regard to organ systems and disease. Performing exhaustive literature searches, we have manually annotated a subset of these 3020 proteins that have cardiovascular-related functions on the basis of an existing body of published information. These cardiovascular-related proteins can be organized into eight groups: markers of inflammation and/or cardiovascular disease, vascular and coagulation, signaling, growth and differentiation, cytoskeletal, transcription factors, channels/receptors and heart failure and remodeling. In addition, analysis of the peptide per protein ratio for MS/MS identification reveals group-specific trends. These findings serve as a resource to interrogate the functions of plasma proteins, and moreover, the list of cardiovascular-related proteins in plasma constitutes a baseline proteomic blueprint for the future development of biosignatures for diseases such as myocardial ischemia and atherosclerosis. PMID:16052623

  1. Proteome reference maps of the Lotus japonicus nodule and root.

    PubMed

    Dam, Svend; Dyrlund, Thomas F; Ussatjuk, Anna; Jochimsen, Bjarne; Nielsen, Kasper; Goffard, Nicolas; Ventosa, Miguel; Lorentzen, Andrea; Gupta, Vikas; Andersen, Stig U; Enghild, Jan J; Ronson, Clive W; Roepstorff, Peter; Stougaard, Jens

    2014-02-01

    Legume symbiosis with rhizobia results in the formation of a specialized organ, the root nodule, where atmospheric dinitrogen is reduced to ammonia. In Lotus japonicus (Lotus), several genes involved in nodule development or nodule function have been defined using biochemistry, genetic approaches, and high-throughput transcriptomics. We have employed proteomics to further understand nodule development. Two developmental stages representing nodules prior to nitrogen fixation (white) and mature nitrogen fixing nodules (red) were compared with roots. In addition, the proteome of a spontaneous nodule formation mutant (snf1) was determined. From nodules and roots, 780 and 790 protein spots from 2D gels were identified and approximately 45% of the corresponding unique gene accessions were common. Including a previous proteomics set from Lotus pod and seed, the common gene accessions were decreased to 7%. Interestingly, an indication of more pronounced PTMs in nodules than in roots was determined. Between the two nodule developmental stages, higher levels of pathogen-related 10 proteins, HSPs, and proteins involved in redox processes were found in white nodules, suggesting a higher stress level at this developmental stage. In contrast, protein spots corresponding to nodulins such as leghemoglobin, asparagine synthetase, sucrose synthase, and glutamine synthetase were prevalent in red nodules. The distinct biochemical state of nodules was further highlighted by the conspicuous presence of several nitrilases, ascorbate metabolic enzymes, and putative rhizobial effectors.

  2. Novel Effects of Hormonal Contraceptive Use on the Plasma Proteome

    PubMed Central

    Fischer, Karina; El-Sohemy, Ahmed

    2012-01-01

    Background Hormonal contraceptive (HC) use may increase cardiometabolic risk; however, the effect of HC on emerging cardiometabolic and other disease risk factors is not clear. Objectives To determine the association between HC use and plasma proteins involved in established and emerging disease risk pathways. Method Concentrations of 54 high-abundance plasma proteins were measured simultaneously by LC-MRM/MS in 783 women from the Toronto Nutrigenomics and Health Study. C-reactive protein (CRP) was measured separately. ANCOVA was used to test differences in protein concentrations between users and non-users, and among HC users depending on total hormone dose. Linear regression was used to test the association between duration (years) of HC use and plasma protein concentrations. Principal components analysis (PCA) was used to identify plasma proteomic profiles in users and non-users. Results After Bonferroni correction, 19 proteins involved in inflammation, innate immunity, coagulation and blood pressure regulation were significantly different between users and non-users (P<0.0009). These differences were replicated across three distinct ethnocultural groups. Traditional markers of glucose and lipid metabolism were also significantly higher among HC users. Neither hormone dose nor duration of use affected protein concentrations. PCA identified 4 distinct proteomic profiles in users and 3 in non-users. Conclusion HC use was associated with different concentrations of plasma proteins along various disease-related pathways, and these differences were present across different ethnicities. Aside from the known effect of HC on traditional biomarkers of cardiometabolic risk, HC use also affects numerous proteins that may be biomarkers of dysregulation in inflammation, coagulation and blood pressure. PMID:22984625

  3. 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.

  4. Seminal plasma proteome of electroejaculated Bos indicus bulls.

    PubMed

    Rego, J P A; Crisp, J M; Moura, A A; Nouwens, A S; Li, Y; Venus, B; Corbet, N J; Corbet, D H; Burns, B M; Boe-Hansen, G B; McGowan, M R

    2014-07-01

    The present study describes the seminal plasma proteome of Bos indicus bulls. Fifty-six, 24-month old Australian Brahman sires were evaluated and subjected to electroejaculation. Seminal plasma proteins were separated by 2-D SDS-PAGE and identified by mass spectrometry. The percentage of progressively motile and morphologically normal sperm of the bulls were 70.4 ± 2.3 and 64 ± 3.2%, respectively. A total of 108 spots were identified in the 2-D maps, corresponding to 46 proteins. Binder of sperm proteins accounted for 55.8% of all spots detected in the maps and spermadhesins comprised the second most abundant constituents. Other proteins of the Bos indicus seminal plasma include clusterin, albumin, transferrin, metalloproteinase inhibitor 2, osteopontin, epididymal secretory protein E1, apolipoprotein A-1, heat shock 70 kDa protein, glutathione peroxidase 3, cathelicidins, alpha-enolase, tripeptidyl-peptidase 1, zinc-alpha-2-glycoprotein, plasma serine protease inhibitor, beta 2-microglobulin, proteasome subunit beta type-4, actin, cathepsins, nucleobinding-1, protein S100-A9, hemoglobin subunit alpha, cadherin-1, angiogenin-1, fibrinogen alpha and beta chain, ephirin-A1, protein DJ-1, serpin A3-7, alpha-2-macroglobulin, annexin A1, complement factor B, polymeric immunoglobulin receptor, seminal ribonuclease, ribonuclease-4, prostaglandin-H2 d-isomerase, platelet-activating factor acetylhydrolase, and phosphoglycerate kinase 1. In conclusion, this work uniquely portrays the Bos indicus seminal fluid proteome, based on samples from a large set of animals representing the Brahman cattle of the tropical Northern Australia. Based on putative biochemical attributes, seminal proteins act during sperm maturation, protection, capacitation and fertilization. PMID:24889044

  5. Reference map for liquid chromatography-mass spectrometry-based quantitative proteomics.

    PubMed

    Kim, Yeoun Jin; Feild, Brian; Fitzhugh, William; Heidbrink, Jenny L; Duff, James W; Heil, Jeremy; Ruben, Steven M; He, Tao

    2009-10-15

    The accurate mass and time (AMT) tag strategy has been recognized as a powerful tool for high-throughput analysis in liquid chromatography-mass spectrometry (LC-MS)-based proteomics. Due to the complexity of the human proteome, this strategy requires highly accurate mass measurements for confident identifications. We have developed a method of building a reference map that allows relaxed criteria for mass errors yet delivers high confidence for peptide identifications. The samples used for generating the peptide database were produced by collecting cysteine-containing peptides from T47D cells and then fractionating the peptides using strong cationic exchange chromatography (SCX). LC-tandem mass spectrometry (MS/MS) data from the SCX fractions were combined to create a comprehensive reference map. After the reference map was built, it was possible to skip the SCX step in further proteomic analyses. We found that the reference-driven identification increases the overall throughput and proteomic coverage by identifying peptides with low intensity or complex interference. The use of the reference map also facilitates the quantitation process by allowing extraction of peptide intensities of interest and incorporating models of theoretical isotope distribution.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  7. 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

  8. 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

  9. Plasma Proteomics, The Human Proteome Project, and Cancer-Associated Alternative Splice Variant Proteins☆, ☆☆

    PubMed Central

    Omenn, Gilbert S.

    2016-01-01

    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. PMID:24211518

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    PubMed Central

    Wickwire, Kathie; Ho, Emily; Chung, Carolyn S.; King, Janet

    2014-01-01

    Zinc (Zn) deficiency is a problem worldwide. 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 immuno-affinity column. An unnamed protein that was related to immunoglobulins was observed in the immunode-pleted 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. PMID:23255060

  12. 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.

  13. 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. PMID:23255060

  14. 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.

  15. Age- and sex-associated plasma proteomic changes in growth hormone receptor gene-disrupted mice.

    PubMed

    Ding, Juan; Berryman, Darlene E; Jara, Adam; Kopchick, John J

    2012-08-01

    Growth hormone receptor gene-disrupted (GHR-/-) mice are dwarf, insulin sensitive, and long lived despite being obese. In order to identify characteristics associated with their increased longevity, we studied age-related plasma proteomic changes in these mice. Male and female GHR-/- mice and their littermate controls were followed longitudinally at 8, 16, and 24 months of ages for plasma proteomic analysis. Relative to control littermates, GHR-/- mice had increased levels of apolipoprotein A-4 and retinol-binding protein-4 and decreased levels of apolipoprotein E, haptoglobin, and mannose-binding protein-C. Female GHR-/- mice showed decreased inflammatory cytokines including interleukin-1β and monocyte chemotactic protein-1. Additionally, sex differences were found in specific isoforms of apolipoprotein E, RBP-4, haptoglobin, albumin, and hemoglobin subunit beta. In conclusion, we find plasma proteomic changes in GHR-/- mice that favor a longer life span as well as sex differences indicative of an improved health span in female mice. PMID:22156438

  16. Plasma proteomic analysis of active and torpid greater mouse-eared bats (Myotis myotis)

    PubMed Central

    Hecht, Alexander M.; Braun, Beate C.; Krause, Eberhard; Voigt, Christian C.; Greenwood, Alex D.; Czirják, Gábor Á.

    2015-01-01

    Hibernation is a physiological adaptation to overcome extreme environmental conditions. It is characterized by prolonged periods of torpor interrupted by temporary arousals during winter. During torpor, body functions are suppressed and restored rapidly to almost pre-hibernation levels during arousal. Although molecular studies have been performed on hibernating rodents and bears, it is unclear how generalizable the results are among hibernating species with different physiology such as bats. As targeted blood proteomic analysis are lacking in small hibernators, we investigated the general plasma proteomic profile of European Myotis myotis and hibernation associated changes between torpid and active individuals by two-dimensional gel electrophoresis. Results revealed an alternation of proteins involved in transport, fuel switching, innate immunity and blood coagulation between the two physiological states. The results suggest that metabolic changes during hibernation are associated with plasma proteomic changes. Further characterization of the proteomic plasma profile identified transport proteins, coagulation proteins and complement factors and detected a high abundance of alpha-fetoprotein. We were able to establish for the first time a basic myotid bat plasma proteomic profile and further demonstrated a modulated protein expression during torpor in Myotis myotis, indicating both novel physiological pathways in bats in general, and during hibernation in particular. PMID:26586174

  17. Plasma proteomic analysis of active and torpid greater mouse-eared bats (Myotis myotis).

    PubMed

    Hecht, Alexander M; Braun, Beate C; Krause, Eberhard; Voigt, Christian C; Greenwood, Alex D; Czirják, Gábor Á

    2015-11-20

    Hibernation is a physiological adaptation to overcome extreme environmental conditions. It is characterized by prolonged periods of torpor interrupted by temporary arousals during winter. During torpor, body functions are suppressed and restored rapidly to almost pre-hibernation levels during arousal. Although molecular studies have been performed on hibernating rodents and bears, it is unclear how generalizable the results are among hibernating species with different physiology such as bats. As targeted blood proteomic analysis are lacking in small hibernators, we investigated the general plasma proteomic profile of European Myotis myotis and hibernation associated changes between torpid and active individuals by two-dimensional gel electrophoresis. Results revealed an alternation of proteins involved in transport, fuel switching, innate immunity and blood coagulation between the two physiological states. The results suggest that metabolic changes during hibernation are associated with plasma proteomic changes. Further characterization of the proteomic plasma profile identified transport proteins, coagulation proteins and complement factors and detected a high abundance of alpha-fetoprotein. We were able to establish for the first time a basic myotid bat plasma proteomic profile and further demonstrated a modulated protein expression during torpor in Myotis myotis, indicating both novel physiological pathways in bats in general, and during hibernation in particular.

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

    PubMed

    Uvackova, Lubica; Ondruskova, Emilia; Danchenko, Maksym; Skultety, Ludovit; Miernyk, Ján A; Hrubík, Pavel; Hajduch, Martin

    2014-11-26

    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. Proteins were isolated from fully developed mature leaves in biological triplicate and analyzed by two-dimensional electrophoresis plus tandem mass spectrometry. Using this approach, we were able to reproducibly quantify 190 abundant protein spots, from which 157 proteins were identified. Most of identified proteins are associated with the energy and protein destination/storage categories. The reference map provides a basis for understanding the accumulation of flavonoids and other phenolic compounds in mature leaves (e.g., identification of chalcone synthase, the first committed enzyme in flavonoid biosynthesis). We additionally detected several proteins of as yet unknown function. These proteins comprise a pool of potential targets that might be useful in nontraditional medical applications. PMID:25365400

  19. Proteomics

    SciTech Connect

    Hixson, Kim K.; Lopez-Ferrer, Daniel; Robinson, Errol W.; Pasa-Tolic, Ljiljana

    2010-02-01

    Proteomics aims to characterize the spatial distribution and temporal dynamics of proteins in biological systems, the protein response to environmental stimuli, and the differences in protein states between diseased and control biological systems. Mass spectrometry (MS) plays a crucial role in enabling the analysis of proteomes and typically is the method of choice for identifying proteins present in biological systems. Peptide (and consequently protein) identifications are made by comparing measured masses to calculated values obtained from genome data. Several methodologies based on MS have been developed for the analysis of proteomes. The complexity of the biological systems requires that the proteome be separated prior to analysis. Both gel based and liquid chromatography based separations have proven very useful in this regard. Typically, separated proteins are analyzed with MS either intact (top-down proteomics) or are digested into peptides (bottom-up) prior to MS analysis. Additionally, several procedures, with and without stable isotopic labeling, have been introduced to facilitate protein quantitation (e.g. characterize changes in protein abundances between given biological states).

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. 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

  7. Two-dimensional proteome reference map of Rhizobium tropici PRF 81 reveals several symbiotic determinants and strong resemblance with agrobacteria.

    PubMed

    Gomes, Douglas Fabiano; Batista, Jesiane Stefania da Silva; Torres, Adalgisa Ribeiro; de Souza Andrade, Diva; Galli-Terasawa, Lygia Vitoria; Hungria, Mariangela

    2012-03-01

    Rhizobium tropici strain PRF 81 is used in commercial inoculants for common-bean crops in Brazil because of its high efficiency in nitrogen fixation and, as in other strains belonging to this species, its tolerance of environmental stresses, representing a useful biological alternative to chemical nitrogen fertilizers. In this study, a proteomic reference map of PRF 81 was obtained by two-dimensional gel electrophoresis and MALDI-TOF/TOF-TOF mass spectrometry. In total, 115 spots representing 109 different proteins were successfully identified, contributing to a better understanding of the rhizobia-legume symbiosis and supporting, at proteomics level, a strong resemblance with agrobacteria.

  8. Comparative proteomic analysis of plasma proteins in patients with age-related macular degeneration

    PubMed Central

    Xu, Xin-Rong; Zhong, Lu; Huang, Bing-Lin; Wei, Yuan-Hua; Zhou, Xin; Wang, Ling; Wang, Fu-Qiang

    2014-01-01

    AIM To find the significant altered proteins in age-related macular degeneration (AMD) patients as potential biomarkers of AMD. METHODS A comparative analysis of the protein pattern of AMD patients versus healthy controls was performed by means of proteomic analysis using two-dimensional gel electrophoresis followed by protein identification with MALDI TOF/TOF mass spectrometry. RESULTS We identified 28 proteins that were significantly altered with clinical relevance in AMD patients. These proteins were involved in a wide range of biological functions including immune responses, growth cytokines, cell fate determination, wound healing, metabolism, and anti-oxidance. CONCLUSION These results demonstrate the capacity of proteomic analysis of AMD patient plasma. In addition to the utility of this approach for biomarker discovery, identification of alterations in endogenous proteins in the plasma of AMD patient could improve our understanding of the disease pathogenesis. PMID:24790867

  9. Effect of increased testicular temperature on seminal plasma proteome of the ram.

    PubMed

    Rocha, David R; Martins, Jorge André M; van Tilburg, Mauricio F; Oliveira, Rodrigo V; Moreno, Frederico B; Monteiro-Moreira, Ana Cristina O; Moreira, Renato A; Araújo, Airton A; Moura, Arlindo A

    2015-11-01

    The present study evaluated the effects of heat stress on the ram seminal plasma proteome. Six Morada Nova rams were scrotal insulated for 8 days. Scrotal circumference, sperm parameters, and seminal fluid proteins were evaluated before (Day 0) and twice during scrotal insulation (Days 4 and 8), and weekly until semen parameters returned to preinsulation values (normal). Seminal proteins were analyzed by two-dimensional SDS-PAGE and mass spectrometry. Scrotal circumference decreased from 30 ± 0.4 cm on Day 0 to 22.6 ± 0.6 cm on Day 36 (P < 0.05) and became equivalent to preinsulation values on Day 71. Motile sperm became nearly absent from Day 8 to Day 64 but returned to normal on Day 113. Percentage of normal sperm changed similarly and returned to normal on Day 106. Rams were azoospermic between Days 29 and 64, and sperm concentration came back to normal on Day 92. The number of spots/two-dimensional gel reduced from 256 ± 31 on Day 0 to 104 ± 14 on Day 29 (when rams were azoospermic) and then increased to 183 ± 9 on Day 113 (P < 0.05), similar to spot counts before insulation. The intensities of 24 spots, referring to 17 seminal plasma proteins, were affected by treatment (P < 0.05). After insulation, seminal plasma had greater expression of actin (two isoforms), albumin, heat shock protein 70 kDa, protein DJ-1, HRPE773-like, C-reactive protein precursor, bodhesin-2 (one isoform), spermadhesins. Most protein spots had the greatest intensity between Days 8 and 29, returning to preinsulation values on Day 113 (when many sperm criteria returned to normal). Proteins downregulated after scrotal insulation included dipeptidyl peptidase 3, isoforms of heat shock protein 90 kDa, RSVP22, MMP2 and of Bdh2. In this case, RSVP22 was reduced on Day 113 and all others, on Day 134. Expression of MMP2 and HSP90.1 was reduced throughout the study. Integrin β5, V-type H(+)-ATPase subunit A, ZBTB 42-like protein, isoforms of Bdh2, PSP-I, and RSVP22 were

  10. Effect of increased testicular temperature on seminal plasma proteome of the ram.

    PubMed

    Rocha, David R; Martins, Jorge André M; van Tilburg, Mauricio F; Oliveira, Rodrigo V; Moreno, Frederico B; Monteiro-Moreira, Ana Cristina O; Moreira, Renato A; Araújo, Airton A; Moura, Arlindo A

    2015-11-01

    The present study evaluated the effects of heat stress on the ram seminal plasma proteome. Six Morada Nova rams were scrotal insulated for 8 days. Scrotal circumference, sperm parameters, and seminal fluid proteins were evaluated before (Day 0) and twice during scrotal insulation (Days 4 and 8), and weekly until semen parameters returned to preinsulation values (normal). Seminal proteins were analyzed by two-dimensional SDS-PAGE and mass spectrometry. Scrotal circumference decreased from 30 ± 0.4 cm on Day 0 to 22.6 ± 0.6 cm on Day 36 (P < 0.05) and became equivalent to preinsulation values on Day 71. Motile sperm became nearly absent from Day 8 to Day 64 but returned to normal on Day 113. Percentage of normal sperm changed similarly and returned to normal on Day 106. Rams were azoospermic between Days 29 and 64, and sperm concentration came back to normal on Day 92. The number of spots/two-dimensional gel reduced from 256 ± 31 on Day 0 to 104 ± 14 on Day 29 (when rams were azoospermic) and then increased to 183 ± 9 on Day 113 (P < 0.05), similar to spot counts before insulation. The intensities of 24 spots, referring to 17 seminal plasma proteins, were affected by treatment (P < 0.05). After insulation, seminal plasma had greater expression of actin (two isoforms), albumin, heat shock protein 70 kDa, protein DJ-1, HRPE773-like, C-reactive protein precursor, bodhesin-2 (one isoform), spermadhesins. Most protein spots had the greatest intensity between Days 8 and 29, returning to preinsulation values on Day 113 (when many sperm criteria returned to normal). Proteins downregulated after scrotal insulation included dipeptidyl peptidase 3, isoforms of heat shock protein 90 kDa, RSVP22, MMP2 and of Bdh2. In this case, RSVP22 was reduced on Day 113 and all others, on Day 134. Expression of MMP2 and HSP90.1 was reduced throughout the study. Integrin β5, V-type H(+)-ATPase subunit A, ZBTB 42-like protein, isoforms of Bdh2, PSP-I, and RSVP22 were

  11. 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

  12. Optimizing Performance of Glycopeptide Capture for Plasma Proteomics

    PubMed Central

    Berven, Frode S.; Ahmad, Rushdy; Clauser, Karl R.; Carr, Steven A.

    2010-01-01

    Selective capture of glycopolypeptides followed by release and analysis of the former glycosylation-site peptides has been shown to have promise for reducing complexity of body fluids such as blood for biomarker discovery. In this work, a protocol based on capture of polypeptides containing a N-linked carbohydrate from human plasma using commercially available magnetic beads coupled with hydrazide chemistry was optimized and partially automated through the use of a KingFisher magnetic particle processor. Comparison of bead-based glycocapture at the protein-level vs. peptide-level revealed differences in the specificity, reproducibility and absolute number of former glycosylation-site peptides detected. Evaluation of a range of capture and elution conditions led to an optimized protocol with a 24% intraday and 30% interday CV, and a glycopeptide capture specificity of 99%. Depleting the plasma of 14 high abundance proteins improved detection sensitivity by approximately one order of magnitude compared to non-depleted plasma and resulted in an increase of 24% in the number of identified glycoproteins. The sensitivity of SPEG for detection of glycoproteins in depleted, non-fractionated plasma was found to be in the 10 to 100 pmol/ml range corresponding to glycoprotein levels ranging from 100’s of nanograms/ml to 10’s of micrograms/ml. Despite high capture specificity,the total number of glycoproteins detected and the sensitivity of SPEG in plasma is surprisingly limited. PMID:20235580

  13. 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

  14. Proteomic methodological recommendations for studies involving human plasma, platelets, and peripheral blood mononuclear cells.

    PubMed

    de Roos, Baukje; Duthie, Susan J; Polley, Abigael C J; Mulholland, Francis; Bouwman, Freek G; Heim, Carolin; Rucklidge, Garry J; Johnson, Ian T; Mariman, Edwin C; Daniel, Hannelore; Elliott, Ruan M

    2008-06-01

    This study was designed to develop, optimize and validate protocols for blood processing prior to proteomic analysis of plasma, platelets and peripheral blood mononuclear cells (PBMC) and to determine analytical variation of a single sample of depleted plasma, platelet and PBMC proteins within and between four laboratories each using their own standard operating protocols for 2D gel electrophoresis. Plasma depleted either using the Beckman Coulter IgY-12 proteome partitioning kit or the Amersham albumin and IgG depletion columns gave good quality gels, but reproducibility appeared better with the single-use immuno-affinity column. The use of the Millipore Filter Device for protein concentration gave a 16% ( p < 0.005) higher recovery of protein in flow-through sample compared with acetone precipitation. The use of OptiPrep gave the lowest level of platelet contamination (1:0.8) during the isolation of PBMC from blood. Several proteins (among which are alpha-tropomyosin, fibrinogen and coagulation factor XIII A) were identified that may be used as biomarkers of platelet contamination in future studies. When identifying preselected spots, at least three out of the four centers found similar identities for 10 out of the 10 plasma proteins, 8 out of the 10 platelet proteins and 8 out of the 10 PBMC proteins. The discrepancy in spot identifications has been described before and may be explained by the mis-selection of spots due to laboratory-to-laboratory variation in gel formats, low scores on the peptide analysis leading to no or only tentative identifications, or incomplete resolution of different proteins in what appears as a single abundant spot. The average within-laboratory coefficient of variation (CV) for each of the matched spots after automatic matching using either PDQuest or ProteomWeaver software ranged between 18 and 69% for depleted plasma proteins, between 21 and 55% for platelet proteins, and between 22 and 38% for PBMC proteins. Subsequent manual

  15. 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

  16. 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. PMID:27196271

  17. 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. PMID:26248320

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  20. Global Stability of Plasma Proteomes for Mass Spectrometry-Based Analyses*

    PubMed Central

    Zimmerman, Lisa J.; Li, Ming; Yarbrough, Wendell G.; Slebos, Robbert J. C.; Liebler, Daniel C.

    2012-01-01

    Peptide-based mass spectrometry approaches, such as multiple reaction monitoring, provide a powerful means to measure candidate protein biomarkers in plasma. A potential confounding problem is the effect of preanalytical variables, which may affect the integrity of proteins and peptides. Although some blood proteins undergo rapid physiological proteolysis ex vivo, the stability of most plasma proteins to preanalytical variables remains largely unexplored. We applied liquid chromatography-tandem mass spectrometry shotgun proteomics and multiple reaction monitoring analyses to characterize the stability of proteins at the peptide level in plasma. We systematically evaluated the effects of delay in plasma preparation at different temperatures, multiple freeze-thaw cycles and erythocyte hemolysis on peptide and protein inventories in prospectively collected human plasma. Time course studies indicated few significant changes in peptide and protein identifications, semitryptic peptides and methionine-oxidized peptides in plasma from blood collected in EDTA plasma tubes and stored for up to a week at 4 °C or room temperature prior to plasma isolation. Similarly, few significant changes were observed in similar analyses of plasma subjected to up to 25 freeze-thaw cycles. Hemolyzed samples produced no significant differences beyond the presence of hemoglobin proteins. Finally, paired comparisons of plasma and serum samples prepared from the same patients also yielded few significant differences, except for the depletion of fibrinogen in serum. Blood proteins thus are broadly stable to preanalytical variables when analyzed at the peptide level. Collection protocols to generate plasma for multiple reaction monitoring-based analyses may have different requirements than for other analyses directed at intact proteins. PMID:22301387

  1. Systems analysis of endothelial cell plasma membrane proteome of rat lung microvasculature

    PubMed Central

    2011-01-01

    Background Endothelial cells line all blood vessels to form the blood-tissue interface which is critical for maintaining organ homeostasis and facilitates molecular exchange. We recently used tissue subcellular fractionation combined with several multi-dimensional mass spectrometry-based techniques to enhance identification of lipid-embedded proteins for large-scale proteomic mapping of luminal endothelial cell plasma membranes isolated directly from rat lungs in vivo. The biological processes and functions of the proteins expressed at this important blood-tissue interface remain unexplored at a large scale. Results We performed an unbiased systems analysis of the endothelial cell surface proteome containing over 1800 proteins to unravel the major functions and pathways apparent at this interface. As expected, many key functions of plasma membranes in general (i.e., cell surface signaling pathways, cytoskeletal organization, adhesion, membrane trafficking, metabolism, mechanotransduction, membrane fusion, and vesicle-mediated transport) and endothelial cells in particular (i.e., blood vessel development and maturation, angiogenesis, regulation of endothelial cell proliferation, protease activity, and endocytosis) were significantly overrepresented in this proteome. We found that endothelial cells express multiple proteins that mediate processes previously reported to be restricted to neuronal cells, such as neuronal survival and plasticity, axon growth and regeneration, synaptic vesicle trafficking and neurotransmitter metabolic process. Surprisingly, molecular machinery for protein synthesis was also detected as overrepresented, suggesting that endothelial cells, like neurons, can synthesize proteins locally at the cell surface. Conclusion Our unbiased systems analysis has led to the potential discovery of unexpected functions in normal endothelium. The discovery of the existence of protein synthesis at the plasma membrane in endothelial cells provides new insight

  2. 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.

  3. 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

  4. Proteomic analysis of white and yellow seminal plasma in turkeys (Meleagris gallopavo).

    PubMed

    Słowińska, M; Kozłowski, K; Jankowski, J; Ciereszko, A

    2015-06-01

    Yellow semen syndrome (YSS) is endemic within domestic turkey populations. Yellow semen is of lower quality and, when used for insemination, results in reduced fertility and hatchability. Little is known about the etiology of YSS. The aim of this study was to compare the proteome of white and yellow seminal plasma of turkeys using 1) 2-dimensional difference gel electrophoresis (2D-DIGE) to quantify seminal plasma proteins and 2) matrix-assisted laser desorption/ionization mass spectrometry to identify the proteins that are differentially abundant in white and yellow seminal plasma. A total of 49 protein spots (30 upregulated and 19 downregulated) were differentially expressed in yellow seminal plasma compared with white seminal plasma. Transthyretin and serum albumin-like showed a 3-fold increase in seminal plasma from males with YSS, and the latter was validated using Western blot analysis. A 3-fold increase was observed for hemopexin-like and immunoglobulin light chain V-J-C region. Pantetheinase-like showed a 1.3-fold increase. Ovotransferrin, hepatocyte growth factor activator, cysteine-rich secretory protein 3-like, and ferritin heavy chain-like showed a significant decrease (at least a 1.3-fold decrease) in yellow semen. Further studies are necessary to evaluate the precise function of the above-mentioned proteins in YSS and to establish quality markers of turkey semen to predict the reproductive potential of individual turkeys.

  5. Proteomic Identification of Novel Differentiation Plasma Protein Markers in Hypobaric Hypoxia-Induced Rat Model

    PubMed Central

    Ahmad, Mohammad Faiz; Sharma, Manish; Garg, Iti; Bhargava, Kalpana

    2014-01-01

    Background Hypobaric hypoxia causes complex changes in the expression of genes, including stress related genes and corresponding proteins that are necessary to maintain homeostasis. Whereas most prior studies focused on single proteins, newer methods allowing the simultaneous study of many proteins could lead to a better understanding of complex and dynamic changes that occur during the hypobaric hypoxia. Methods In this study we investigated the temporal plasma protein alterations of rat induced by hypobaric hypoxia at a simulated altitude of 7620 m (25,000 ft, 282 mm Hg) in a hypobaric chamber. Total plasma proteins collected at different time points (0, 6, 12 and 24 h), separated by two-dimensional electrophoresis (2-DE) and identified using matrix assisted laser desorption ionization time of flight (MALDI-TOF/TOF). Biological processes that were enriched in the plasma proteins during hypobaric hypoxia were identified using Gene Ontology (GO) analysis. According to their properties and obvious alterations during hypobaric hypoxia, changes of plasma concentrations of Ttr, Prdx-2, Gpx -3, Apo A-I, Hp, Apo-E, Fetub and Nme were selected to be validated by Western blot analysis. Results Bioinformatics analysis of 25 differentially expressed proteins showed that 23 had corresponding candidates in the database. The expression patterns of the eight selected proteins observed by Western blot were in agreement with 2-DE results, thus confirming the reliability of the proteomic analysis. Most of the proteins identified are related to cellular defense mechanisms involving anti-inflammatory and antioxidant activity. Their presence reflects the consequence of serial cascades initiated by hypobaric hypoxia. Conclusion/Significance This study provides information about the plasma proteome changes induced in response to hypobaric hypoxia and thus identification of the candidate proteins which can act as novel biomarkers. PMID:24842778

  6. 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

  7. 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.

  8. Affinity proteomic profiling of plasma, cerebrospinal fluid, and brain tissue within multiple sclerosis.

    PubMed

    Byström, Sanna; Ayoglu, Burcu; Häggmark, Anna; Mitsios, Nicholas; Hong, Mun-Gwan; Drobin, Kimi; Forsström, Björn; Fredolini, Claudia; Khademi, Mohsen; Amor, Sandra; Uhlén, Mathias; Olsson, Tomas; Mulder, Jan; Nilsson, Peter; Schwenk, Jochen M

    2014-11-01

    The brain is a vital organ and because it is well shielded from the outside environment, possibilities for noninvasive analysis are often limited. Instead, fluids taken from the spinal cord or circulatory system are preferred sources for the discovery of candidate markers within neurological diseases. In the context of multiple sclerosis (MS), we applied an affinity proteomic strategy and screened 22 plasma samples with 4595 antibodies (3450 genes) on bead arrays, then defined 375 antibodies (334 genes) for targeted analysis in a set of 172 samples and finally used 101 antibodies (43 genes) on 443 plasma as well as 573 cerebrospinal spinal fluid (CSF) samples. This revealed alteration of protein profiles in relation to MS subtypes for IRF8, IL7, METTL14, SLC30A7, and GAP43. Respective antibodies were subsequently used for immunofluorescence on human post-mortem brain tissue with MS pathology for expression and association analysis. There, antibodies for IRF8, IL7, and METTL14 stained neurons in proximity of lesions, which highlighted these candidate protein targets for further studies within MS and brain tissue. The affinity proteomic translation of profiles discovered by profiling human body fluids and tissue provides a powerful strategy to suggest additional candidates to studies of neurological disorders.

  9. 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

  10. Plasma proteome analysis reveals the geographical origin and liver tumor status of Dab (Limanda limanda) from UK marine waters.

    PubMed

    Ward, Douglas G; Wei, Wenbin; Cheng, Yaping; Billingham, Lucinda J; Martin, Ashley; Johnson, Philip I; Lyons, Brett P; Feist, Stephen W; Stentiford, Grant D

    2006-06-15

    The flatfish species dab (Limanda limanda) is the sentinel for offshore marine monitoring in the United Kingdom National Marine Monitoring Programme (NMMP). At certain sites in the North and Irish Seas, the prevalence of macroscopic liver tumors can exceed 10%. The plasma proteome of these fish potentially contains reporter proteins or "biomarkers" that may enable development of diagnostic tests for liver cancer and further our understanding of the disease. Following selection of sample groups by quality-assured histopathology ("phenotype anchoring"), we used surface-enhanced laser desorption/ionization (SELDI) time-of-flight mass spectrometry to produce proteomic profiles of plasma from 213 dab collected during the 2004 UK NMMP. The resulting protein profiles were compared between fish from the North and Irish Seas and between fish with liver neoplasia or nondiseased liver. Significant differences were found between the plasma proteomes of dab from the North Sea and Irish Sea, which in conjunction with artificial neural networks can correctly determine from which sea dab were captured in 85% of the cases. In addition, the presence of liver tumors is associated with significant changes in the plasma proteome. We conclude that SELDI-based plasma profiling is potentially of use in nonlethal marine monitoring using wild sentinels such as dab. Furthermore, accurate selection of sample groups is critical for avoiding effects of confounding factors such as age, gender, and geographic origin of samples. PMID:16830578

  11. Shotgun proteomics and network analysis between plasma membrane and extracellular matrix proteins from rat olfactory ensheathing cells.

    PubMed

    Liu, Yisong; Teng, Xiaohua; Yang, Xiaoxu; Song, Qing; Lu, Rong; Xiong, Jixian; Liu, Bo; Zeng, Nianju; Zeng, Yu; Long, Jia; Cao, Rui; Lin, Yong; He, Quanze; Chen, Ping; Lu, Ming; Liang, Songping

    2010-01-01

    Olfactory ensheathing cells (OECs) are a special type of glial cells that have characteristics of both astrocytes and Schwann cells. Evidence suggests that the regenerative capacity of OECs is induced by soluble, secreted factors that influence their microenvironment. These factors may regulate OECs self-renewal and/or induce their capacity to augment spinal cord regeneration. Profiling of plasma membrane and extracellular matrix through a high-throughput expression proteomics approach was undertaken to identify plasma membrane and extracellular matrix proteins of OECs under serum-free conditions. 1D-shotgun proteomics followed with gene ontology (GO) analysis was used to screen proteins from primary culture rat OECs. Four hundred and seventy nonredundant plasma membrane proteins and 168 extracellular matrix proteins were identified, the majority of which were never before reported to be produced by OECs. Furthermore, plasma membrane and extracellular proteins were classified based on their protein-protein interaction predicted by STRING quantitatively integrates interaction data. The proteomic profiling of the OECs plasma membrane proteins and their connection with the secretome in serum-free culture conditions provides new insights into the nature of their in vivo microenvironmental niche. Proteomic analysis for the discovery of clinical biomarkers of OECs mechanism warrants further study.

  12. 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

  13. Plasma Fractionation Enriches Post-Myocardial Infarction Samples Prior to Proteomics Analysis

    PubMed Central

    de Castro Brás, Lisandra E.; DeLeon, Kristine Y.; Ma, Yonggang; Dai, Qiuxia; Hakala, Kevin; Weintraub, Susan T.; Lindsey, Merry L.

    2012-01-01

    Following myocardial infarction (MI), matrix metalloproteinase-9 (MMP-9) levels increase, and MMP-9 deletion improves post-MI remodeling of the left ventricle (LV). We provide here a technical report on plasma-analysis from wild type (WT) and MMP-9 null mice using fractionation and mass-spectrometry-based proteomics. MI was induced by coronary artery ligation in male WT and MMP-9 null mice (4–8 months old; n = 3/genotype). Plasma was collected on days 0 (pre-) and 1 post-MI. Plasma proteins were fractionated and proteins in the lowest (fraction 1) and highest (fraction 12) molecular weight fractions were separated by 1-D SDS-PAGE, digested in-gel with trypsin and analyzed by HPLC-ESI-MS/MS on an Orbitrap Velos. We tried five different fractionation protocols, before reaching an optimized protocol that allowed us to identify over 100 proteins. Serum amyloid A substantially increased post-MI in both genotypes, while alpha-2 macroglobulin increased only in the null samples. In fraction 12, extracellular matrix proteins were observed only post-MI. Interestingly, fibronectin-1, a substrate of MMP-9, was identified at both day 0 and day 1 post-MI in the MMP-9 null mice but was only identified post-MI in the WT mice. In conclusion, plasma fractionation offers an improved depletion-free method to evaluate plasma changes following MI. PMID:22778955

  14. 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

  15. Large-Scale Multiplexed Quantitative Discovery Proteomics Enabled by the Use of an O-18-Labeled “Universal” Reference Sample

    SciTech Connect

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

    2009-01-01

    Quantitative comparison of protein abundances across a relatively large number of patient samples is an important challenge for clinical proteomic applications. Herein we describe a dual-quantitation strategy that allows the simultaneous integration of complementary label-free and stable isotope labeling based approaches without increasing the number of LC-MS analyses. The approach utilizes a stable isotope 18O-labeled “universal” reference sample as a comprehensive set of internal standards spiked into each individually processed unlabeled patient sample. The quantitative data are based on both the direct 16O-MS intensities for label-free quantitation and the 16O/18O isotopic peptide pair ratios that compare each patient sample to the identical labeled reference. The effectiveness of this dual-quantitation approach for large scale quantitative proteomics is demonstrated by the application to a set of 38 clinical plasma samples from surviving and non-surviving severe burn patients. With the coupling of immunoaffinity depletion, cysteinyl-peptide enrichment based fractionation, high resolution LC-MS measurements, and the dual-quantitation approach, a total of 318 proteins were confidently quantified with at least two peptides and 263 proteins were quantified by both approaches. The strategy also enabled a direct comparison between the two approaches with the labeling approach showing significantly better precision in quantitation while the label-free approach resulted in more protein identifications. The relative abundance differences determined by the two approaches also show strong correlation. Finally, the dual-quantitation strategy allowed us to identify more candidate protein biomarkers, illustrating the complementary nature of the two quantitative methods.

  16. With or without you - Proteomics with or without major plasma/serum proteins.

    PubMed

    Gianazza, Elisabetta; Miller, Ingrid; Palazzolo, Luca; Parravicini, Chiara; Eberini, Ivano

    2016-05-17

    The first sections of this review compile and discuss strategies and protocols for managing plasma/serum as a source of biomarkers relevant to human disease. In many such cases, depletion of abundant protein(s) is a crucial preliminary step to the procedure; specific conceptual and technical approaches, however, make it possible to effectively use to this purpose whole plasma/serum. The final sections focus instead on the complexity associated with each of the major serum/plasma proteins in terms of both, multiple molecular structures (existence of a number of protein species) and of multiple molecular functions (behavior as multifunctional/multitasking/moonlighting proteins). Reviewing evidence in these and some related fields (regulation of the synthetic pattern by proteins and non-protein compounds and its connection with health and disease) prompts the suggestion/recommendation that information on the abundant components of plasma/serum proteome is routinely obtained and processed/mined as a valuable contribution to the characterization of any non-physiological condition and to the understanding of its mechanisms and of its implications/sequels. PMID:27072114

  17. 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.

  18. 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

  19. 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

    2016-01-01

    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.

  20. With or without you - Proteomics with or without major plasma/serum proteins.

    PubMed

    Gianazza, Elisabetta; Miller, Ingrid; Palazzolo, Luca; Parravicini, Chiara; Eberini, Ivano

    2016-05-17

    The first sections of this review compile and discuss strategies and protocols for managing plasma/serum as a source of biomarkers relevant to human disease. In many such cases, depletion of abundant protein(s) is a crucial preliminary step to the procedure; specific conceptual and technical approaches, however, make it possible to effectively use to this purpose whole plasma/serum. The final sections focus instead on the complexity associated with each of the major serum/plasma proteins in terms of both, multiple molecular structures (existence of a number of protein species) and of multiple molecular functions (behavior as multifunctional/multitasking/moonlighting proteins). Reviewing evidence in these and some related fields (regulation of the synthetic pattern by proteins and non-protein compounds and its connection with health and disease) prompts the suggestion/recommendation that information on the abundant components of plasma/serum proteome is routinely obtained and processed/mined as a valuable contribution to the characterization of any non-physiological condition and to the understanding of its mechanisms and of its implications/sequels.

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

    PubMed

    Hopff, David; Wienkoop, Stefanie; Lüthje, Sabine

    2013-10-01

    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. PMID:23353019

  2. 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

  3. 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

  4. Discovery of Prognostic Biomarker Candidates of Lacunar Infarction by Quantitative Proteomics of Microvesicles Enriched Plasma

    PubMed Central

    Datta, Arnab; Chen, Christopher P.; Sze, Siu Kwan

    2014-01-01

    Background Lacunar infarction (LACI) is a subtype of acute ischemic stroke affecting around 25% of all ischemic stroke cases. Despite having an excellent recovery during acute phase, certain LACI patients have poor mid- to long-term prognosis due to the recurrence of vascular events or a decline in cognitive functions. Hence, blood-based biomarkers could be complementary prognostic and research tools. Methods and Finding Plasma was collected from forty five patients following a non-disabling LACI along with seventeen matched control subjects. The LACI patients were monitored prospectively for up to five years for the occurrence of adverse outcomes and grouped accordingly (i.e., LACI-no adverse outcome, LACI-recurrent vascular event, and LACI-cognitive decline without any recurrence of vascular events). Microvesicles-enriched fractions isolated from the pooled plasma of four groups were profiled by an iTRAQ-guided discovery approach to quantify the differential proteome. The data have been deposited to the ProteomeXchange with identifier PXD000748. Bioinformatics analysis and data mining revealed up-regulation of brain-specific proteins including myelin basic protein, proteins of coagulation cascade (e.g., fibrinogen alpha chain, fibrinogen beta chain) and focal adhesion (e.g., integrin alpha-IIb, talin-1, and filamin-A) while albumin was down-regulated in both groups of patients with adverse outcome. Conclusion This data set may offer important insight into the mechanisms of poor prognosis and provide candidate prognostic biomarkers for validation on larger cohort of individual LACI patients. PMID:24752076

  5. 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

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

    PubMed

    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

  7. 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.

  8. Evaluation of two cell surface modification methods for proteomic analysis of plasma membrane from isolated mouse hepatocytes.

    PubMed

    Li, Xuanwen; Jin, Qihui; Cao, Jia; Xie, Chunliang; Cao, Rui; Liu, Zhen; Xiong, Jixian; Li, Jianglin; Yang, Xiaoxu; Chen, Ping; Liang, Songping

    2009-01-01

    The hepatocyte is a highly polarized cell with a heterogeneous distribution of plasma-membrane (PM) proteins. To reduce the complexity of the proteome of liver tissue and give a comprehensive profile of hepatocyte PM, two PM purification methods based on cell surface modification, named the biotin-avidin (BA) and cationic silica-polyanion (CSP) strategies were evaluated and compared with the traditional cell fractionation method to prepare highly enriched PM from freshly isolated C57 mouse hepatocytes. Employing different principles for PM modification, both methods were effective in the isolation of general and purified PM fraction. The CSP strategy showed better yield for the PM purification from freshly isolated hepatocytes. 189 non-redundant proteins were identified, including 49 from the BA method and 185 from CSP strategy. Many known and novel PM-associated proteins were also found. Our evaluation here should give implications for PM preparation from other freshly isolated tissue-derived cells. The hepatocyte PM proteins identified here should be taken as a references for the PM-related functional and diseases research.

  9. 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.

  10. A large synthetic peptide and phosphopeptide reference library for mass spectrometry-based proteomics.

    PubMed

    Marx, Harald; Lemeer, Simone; Schliep, Jan Erik; Matheron, Lucrece; Mohammed, Shabaz; Cox, Jürgen; Mann, Matthias; Heck, Albert J R; Kuster, Bernhard

    2013-06-01

    We present a peptide library and data resource of >100,000 synthetic, unmodified peptides and their phosphorylated counterparts with known sequences and phosphorylation sites. Analysis of the library by mass spectrometry yielded a data set that we used to evaluate the merits of different search engines (Mascot and Andromeda) and fragmentation methods (beam-type collision-induced dissociation (HCD) and electron transfer dissociation (ETD)) for peptide identification. We also compared the sensitivities and accuracies of phosphorylation-site localization tools (Mascot Delta Score, PTM score and phosphoRS), and we characterized the chromatographic behavior of peptides in the library. We found that HCD identified more peptides and phosphopeptides than did ETD, that phosphopeptides generally eluted later from reversed-phase columns and were easier to identify than unmodified peptides and that current computational tools for proteomics can still be substantially improved. These peptides and spectra will facilitate the development, evaluation and improvement of experimental and computational proteomic strategies, such as separation techniques and the prediction of retention times and fragmentation patterns.

  11. 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.

  12. 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

  13. 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.

  14. 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. PMID:25622825

  15. 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

    2016-03-01

    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. PMID:26851364

  16. 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.

  17. 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

    2010-01-01

    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.

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

    PubMed

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

    2011-05-01

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

  19. 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

  20. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  2. 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.

  3. Statistical considerations of optimal study design for human plasma proteomics and biomarker discovery.

    PubMed

    Zhou, Cong; Simpson, Kathryn L; Lancashire, Lee J; Walker, Michael J; Dawson, Martin J; Unwin, Richard D; Rembielak, Agata; Price, Patricia; West, Catharine; Dive, Caroline; Whetton, Anthony D

    2012-04-01

    A mass spectrometry-based plasma biomarker discovery workflow was developed to facilitate biomarker discovery. Plasma from either healthy volunteers or patients with pancreatic cancer was 8-plex iTRAQ labeled, fractionated by 2-dimensional reversed phase chromatography and subjected to MALDI ToF/ToF mass spectrometry. Data were processed using a q-value based statistical approach to maximize protein quantification and identification. Technical (between duplicate samples) and biological variance (between and within individuals) were calculated and power analysis was thereby enabled. An a priori power analysis was carried out using samples from healthy volunteers to define sample sizes required for robust biomarker identification. The result was subsequently validated with a post hoc power analysis using a real clinical setting involving pancreatic cancer patients. This demonstrated that six samples per group (e.g., pre- vs post-treatment) may provide sufficient statistical power for most proteins with changes>2 fold. A reference standard allowed direct comparison of protein expression changes between multiple experiments. Analysis of patient plasma prior to treatment identified 29 proteins with significant changes within individual patient. Changes in Peroxiredoxin II levels were confirmed by Western blot. This q-value based statistical approach in combination with reference standard samples can be applied with confidence in the design and execution of clinical studies for predictive, prognostic, and/or pharmacodynamic biomarker discovery. The power analysis provides information required prior to study initiation.

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

    PubMed

    Simmons, Denina B D; Sherry, James P

    2016-09-01

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

  5. Shotgun Proteomic Analysis of Plasma from Dairy Cattle Suffering from Footrot: Characterization of Potential Disease-Associated Factors

    PubMed Central

    Sun, Dongbo; Zhang, Hong; Guo, Donghua; Sun, Anguo; Wang, Hongbin

    2013-01-01

    The plasma proteome of healthy dairy cattle and those with footrot was investigated using a shotgun LC-MS/MS approach. In total, 648 proteins were identified in healthy plasma samples, of which 234 were non-redundant proteins and 123 were high-confidence proteins; 712 proteins were identified from footrot plasma samples, of which 272 were non-redundant proteins and 138 were high-confidence proteins. The high-confidence proteins showed significant differences between healthy and footrot plasma samples in molecular weight, isoelectric points and the Gene Ontology categories. 22 proteins were found that may differentiate between the two sets of plasma proteins, of which 16 potential differential expression (PDE) proteins from footrot plasma involved in immunoglobulins, innate immune recognition molecules, acute phase proteins, regulatory proteins, and cell adhesion and cytoskeletal proteins; 6 PDE proteins from healthy plasma involved in regulatory proteins, cytoskeletal proteins and coagulation factors. Of these PDE proteins, haptoglobin, SERPINA10 protein, afamin precursor, haptoglobin precursor, apolipoprotein D, predicted peptidoglycan recognition protein L (PGRP-L) and keratan sulfate proteoglycan (KS-PG) were suggested to be potential footrot-associated factors. The PDE proteins PGRP-L and KS-PG were highlighted as potential biomarkers of footrot in cattle. The resulting protein lists and potential differentially expressed proteins may provide valuable information to increase understanding of plasma protein profiles in cattle and to assist studies of footrot-associated factors. PMID:23418487

  6. 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.

  7. 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. PMID:27039242

  8. Free-Flow Electrophoresis of Plasma Membrane Vesicles Enriched by Two-Phase Partitioning Enhances the Quality of the Proteome from Arabidopsis Seedlings.

    PubMed

    de Michele, Roberto; McFarlane, Heather E; Parsons, Harriet T; Meents, Miranda J; Lao, Jeemeng; González Fernández-Niño, Susana M; Petzold, Christopher J; Frommer, Wolf B; Samuels, A Lacey; Heazlewood, Joshua L

    2016-03-01

    The plant plasma membrane is the interface between the cell and its environment undertaking a range of important functions related to transport, signaling, cell wall biosynthesis, and secretion. Multiple proteomic studies have attempted to capture the diversity of proteins in the plasma membrane using biochemical fractionation techniques. In this study, two-phase partitioning was combined with free-flow electrophoresis to produce a population of highly purified plasma membrane vesicles that were subsequently characterized by tandem mass spectroscopy. This combined high-quality plasma membrane isolation technique produced a reproducible proteomic library of over 1000 proteins with an extended dynamic range including plasma membrane-associated proteins. The approach enabled the detection of a number of putative plasma membrane proteins not previously identified by other studies, including peripheral membrane proteins. Utilizing multiple data sources, we developed a PM-confidence score to provide a value indicating association to the plasma membrane. This study highlights over 700 proteins that, while seemingly abundant at the plasma membrane, are mostly unstudied. To validate this data set, we selected 14 candidates and transiently localized 13 to the plasma membrane using a fluorescent tag. Given the importance of the plasma membrane, this data set provides a valuable tool to further investigate important proteins. The mass spectrometry data are available via ProteomeXchange, identifier PXD001795.

  9. 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

  10. 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.

  11. Mass spectrometry-based analysis of rat liver and hepatocellular carcinoma Morris hepatoma 7777 plasma membrane proteome.

    PubMed

    Cao, Lulu; Clifton, James G; Reutter, Werner; Josic, Djuro

    2013-09-01

    The gel-based proteomic analysis of plasma membranes from rat liver and chemically induced, malignant hepatocellular carcinoma Morris hepatoma 7777 was systematically optimized to yield the maximum number of proteins containing transmembrane domains (TMDs). Incorporation of plasma membrane proteins into a polyacrylamide "tube gel" followed by in-gel digestion of "tube gel" pieces significantly improved detection by electrospray ionization-liquid chromatography-tandem mass spectrometry. Removal of less hydrophobic proteins by washing isolated plasma membranes with 0.1 M sodium carbonate enables detection of a higher number of hydrophobic proteins containing TMDs in both tissues. Subsequent treatment of plasma membranes by a proteolytic enzyme (trypsin) causes the loss of some of the proteins that are detected after washing with sodium carbonate, but it enables the detection of other hydrophobic proteins containing TMDs. Introduction of mass spectrometers with higher sensitivity, higher mass resolution and mass accuracy, and a faster scan rate significantly improved detection of membrane proteins, but the improved sample preparation is still useful and enables detection of additional hydrophobic proteins. Proteolytic predigestion of plasma membranes enables detection of additional hydrophobic proteins and better sequence coverage of TMD-containing proteins in plasma membranes from both tissues.

  12. 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. PMID:27578514

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

    PubMed Central

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

    2016-01-01

    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. PMID:27578514

  14. A two-dimensional electrophoresis proteomic reference map and systematic identification of 1367 proteins from a cell suspension culture of the model legume Medicago truncatula.

    PubMed

    Lei, Zhentian; Elmer, Aaron M; Watson, Bonnie S; Dixon, Richard A; Mendes, Pedro J; Sumner, Lloyd W

    2005-11-01

    The proteome of a Medicago truncatula cell suspension culture was analyzed using two-dimensional electrophoresis and nanoscale HPLC coupled to a tandem Q-TOF mass spectrometer (QSTAR Pulsar i) to yield an extensive protein reference map. Coomassie Brilliant Blue R-250 was used to visualize more than 1661 proteins, which were excised, subjected to in-gel trypsin digestion, and analyzed using nanoscale HPLC/MS/MS. The resulting spectral data were queried against a custom legume protein database using the MASCOT search engine. A total of 1367 of the 1661 proteins were identified with high rigor, yielding an identification success rate of 83% and 907 unique protein accession numbers. Functional annotation of the M. truncatula suspension cell proteins revealed a complete tricarboxylic acid cycle, a nearly complete glycolytic pathway, a significant portion of the ubiquitin pathway with the associated proteolytic and regulatory complexes, and many enzymes involved in secondary metabolism such as flavonoid/isoflavonoid, chalcone, and lignin biosynthesis. Proteins were also identified from most other functional classes including primary metabolism, energy production, disease/defense, protein destination/storage, protein synthesis, transcription, cell growth/division, and signal transduction. This work represents the most extensive proteomic description of M. truncatula suspension cells to date and provides a reference map for future comparative proteomic and functional genomic studies of the response of these cells to biotic and abiotic stress.

  15. Quantitative Proteomic (iTRAQ) Analysis of 1st Trimester Maternal Plasma Samples in Pregnancies at Risk for Preeclampsia

    PubMed Central

    Kolla, Varaprasad; Jenö, Paul; Moes, Suzette; Lapaire, Olav; Hoesli, Irene; Hahn, Sinuhe

    2012-01-01

    A current major obstacle is that no reliable screening markers exist to detect pregnancies at risk for preeclampsia. Quantitative proteomic analysis employing isobaric labelling (iTRAQ) has been suggested to be suitable for the detection of potential plasma biomarkers, a feature we recently verified in analysis of pregnancies with Down syndrome foetuses. We have now examined whether this approach could yield biomarkers to screen pregnancies at risk for preeclampsia. In our study, we used maternal plasma samples obtained at 12 weeks of gestation, six from women who subsequently developed preeclampsia and six with uncomplicated deliveries. In our analysis, we observed elevations in 10 proteins out of 64 proteins in the preeclampsia study group when compared to the healthy control group. These proteins included clusterin, fibrinogen, fibronectin, and angiotensinogen, increased levels of which are known to be associated with preeclampsia. An elevation in the immune-modulatory molecule, galectin 3 binding protein, was also noted. Our pilot study, therefore, indicates that quantitative proteomic iTRAQ analysis could be a useful tool for the detection of new preeclampsia screening markers. PMID:22570525

  16. 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

  17. Proteomic identification of plasma protein tyrosine phosphatase alpha and fibronectin associated with liver fluke, Opisthorchis viverrini, infection.

    PubMed

    Khoontawad, Jarinya; Laothong, Umawadee; Roytrakul, Sittiruk; Pinlaor, Porntip; Mulvenna, Jason; Wongkham, Chaisiri; Yongvanit, Puangrat; Pairojkul, Chawalit; Mairiang, Eimorn; Sithithaworn, Paiboon; Pinlaor, Somchai

    2012-01-01

    Opisthorchiasis caused by Opisthorchis viverrini induces periductal fibrosis via host immune/inflammatory responses. Plasma protein alteration during host-parasite interaction-mediated inflammation may provide potential diagnostic and/or prognostic biomarkers. To search for target protein changes in O. viverrini-infected hamsters, a 1-D PAGE gel band was trypsin-digested and analyzed by a LC-MS/MS-based proteomics approach in the plasma profile of infected hamsters, and applied to humans. Sixty seven proteins were selected for further analysis based on at least two unique tryptic peptides with protein ID score >10 and increased expression at least two times across time points. These proteins have not been previously identified in O. viverrini-associated infection. Among those, proteins involved in structural (19%), immune response (13%), cell cycle (10%) and transcription (10%) were highly expressed. Western blots revealed an expression level of protein tyrosine phosphatase alpha (PTPα) which reached a peak at 1 month and subsequently tended to decrease. Fibronectin significantly increased at 1 month and tended to increase with time, supporting proteomic analysis. PTPα was expressed in the cytoplasm of inflammatory cells, while fibronectin was observed mainly in the cytoplasm of fibroblasts and the extracellular matrix at periductal fibrosis areas. In addition, these protein levels significantly increased in the plasma of O. viverrini-infected patients compared to healthy individuals, and significantly decreased at 2-months post-treatment, indicating their potential as disease markers. In conclusion, our results suggest that plasma PTPα and fibronectin may be associated with opisthorchiasis and the hamster model provides the basis for development of novel diagnostic markers in the future.

  18. Effect of coenzyme Q10 on proteomic profile of blood plasma and cytosolic and microsomal fractions of rat hepatocytes during ontogeny.

    PubMed

    Sharanova, N E; Toropygin, I Yu; Khriapova, E V; Vasilyev, A V; Gapparov, M M G

    2012-11-01

    The proteomic features of blood plasma and subcellular fractions of rat hepatocytes were studied during long-term dietary consumption of coenzyme Q10 as the endogenous mediator of antioxidant and energy homeostasis in the cell. Long-term coenzyme Q10 consumption was followed by the formation of specific nutriproteomes of the microsomal and cytosolic fractions of rat hepatocytes.

  19. Plasma membrane proteomics in the maize primary root growth zone: novel insights into root growth adaptation to water stress.

    PubMed

    Voothuluru, Priyamvada; Anderson, Jeffrey C; Sharp, Robert E; Peck, Scott C

    2016-09-01

    Previous work on maize (Zea mays L.) primary root growth under water stress showed that cell elongation is maintained in the apical region of the growth zone but progressively inhibited further from the apex. These responses involve spatially differential and coordinated regulation of osmotic adjustment, modification of cell wall extensibility, and other cellular growth processes that are required for root growth under water-stressed conditions. As the interface between the cytoplasm and the apoplast (including the cell wall), the plasma membrane likely plays critical roles in these responses. Using a simplified method for enrichment of plasma membrane proteins, the developmental distribution of plasma membrane proteins was analysed in the growth zone of well-watered and water-stressed maize primary roots. The results identified 432 proteins with differential abundances in well-watered and water-stressed roots. The majority of changes involved region-specific patterns of response, and the identities of the water stress-responsive proteins suggest involvement in diverse biological processes including modification of sugar and nutrient transport, ion homeostasis, lipid metabolism, and cell wall composition. Integration of the distinct, region-specific plasma membrane protein abundance patterns with results from previous physiological, transcriptomic and cell wall proteomic studies reveals novel insights into root growth adaptation to water stress.

  20. Plasma membrane proteomics in the maize primary root growth zone: novel insights into root growth adaptation to water stress.

    PubMed

    Voothuluru, Priyamvada; Anderson, Jeffrey C; Sharp, Robert E; Peck, Scott C

    2016-09-01

    Previous work on maize (Zea mays L.) primary root growth under water stress showed that cell elongation is maintained in the apical region of the growth zone but progressively inhibited further from the apex. These responses involve spatially differential and coordinated regulation of osmotic adjustment, modification of cell wall extensibility, and other cellular growth processes that are required for root growth under water-stressed conditions. As the interface between the cytoplasm and the apoplast (including the cell wall), the plasma membrane likely plays critical roles in these responses. Using a simplified method for enrichment of plasma membrane proteins, the developmental distribution of plasma membrane proteins was analysed in the growth zone of well-watered and water-stressed maize primary roots. The results identified 432 proteins with differential abundances in well-watered and water-stressed roots. The majority of changes involved region-specific patterns of response, and the identities of the water stress-responsive proteins suggest involvement in diverse biological processes including modification of sugar and nutrient transport, ion homeostasis, lipid metabolism, and cell wall composition. Integration of the distinct, region-specific plasma membrane protein abundance patterns with results from previous physiological, transcriptomic and cell wall proteomic studies reveals novel insights into root growth adaptation to water stress. PMID:27341663

  1. Chlamydomonas proteomics.

    PubMed

    Rolland, Norbert; Atteia, Ariane; Decottignies, Paulette; Garin, Jérôme; Hippler, Michael; Kreimer, Georg; Lemaire, Stéphane D; Mittag, Maria; Wagner, Volker

    2009-06-01

    Chlamydomonas reinhardtii is a biflagellate and photosynthetic unicellular alga that has long fascinated scientists because it combines characteristics of both plants and animals. Chlamydomonas offers the simplicity of a unicellular organism that is amenable to genetic screening, molecular, and biochemical approaches, as well as to transformation of its nuclear, plastid, or mitochondrial genomes. Over the past decade, proteomics based studies of Chlamydomonas have provided major research contributions in the areas of photosynthesis, molecular biology, and evolution. This review refers to technical and biological aspects of proteomics studies that have been recently performed on the C. reinhardtii model organism.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. Proteomic identification of alpha-2-HS-glycoprotein as a plasma biomarker of hypopharyngeal squamous cell carcinoma.

    PubMed

    Tian, Wen-Dong; Li, Jun-Zheng; Hu, Shui-Wang; Peng, Xiao-Wei; Li, Gang; Liu, Xiong; Chen, Huai-Hong; Xu, Xia; Li, Xiang-Ping

    2015-01-01

    Hypopharyngeal squamous cell carcinoma (HSCC) has very poor prognosis compared with other head and neck squamous cell carcinomas. Late-stage diagnosis of HSCC increases mortality. Therefore, more effective biomarkers for early diagnosis of HSCC are necessary. Unfortunately, appropriate biomarkers for clinical diagnosis and prognosis have not been identified yet. However, recent progresses in quantitative proteomics have offered opportunities to identify plasma proteins as biomarkers for HSCC. In the present study, plasma samples were analyzed by two-dimensional differential gel electrophoresis (2D-DIGE), and differentially expressed proteins were identified by matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS). A total of 26 proteins representing 12 unique gene products were identified. The up-regulation proteins were alpha-2-HS-glycoprotein (AHSG), complement C4-B, haptoglobin, C-reactive protein, and ceruloplasmin, whereas the down-regulation proteins were serum albumin, angiotensinogen, alpha-1-antichymotrypsin, Ig gamma-3 chain C region, fibrinogen gamma chain, apolipoprotein A-I, and Ig kappa chain C region. Among all the differentially expressed proteins, AHSG was validated by western blot and ELISA. The results were consistent with the data from 2D-DIGE, further suggesting that AHSG may be employed as a potential biomarker for the early diagnosis of HSCC. In summary, this study was the first to use 2D-DIGE and MALDI-TOF/TOF platform to identify the potential plasma biomarkers for HSCC. The plasma AHSG showed great potential for HSCC screening. PMID:26464644

  7. Proteomic identification of alpha-2-HS-glycoprotein as a plasma biomarker of hypopharyngeal squamous cell carcinoma

    PubMed Central

    Tian, Wen-Dong; Li, Jun-Zheng; Hu, Shui-Wang; Peng, Xiao-Wei; Li, Gang; Liu, Xiong; Chen, Huai-Hong; Xu, Xia; Li, Xiang-Ping

    2015-01-01

    Hypopharyngeal squamous cell carcinoma (HSCC) has very poor prognosis compared with other head and neck squamous cell carcinomas. Late-stage diagnosis of HSCC increases mortality. Therefore, more effective biomarkers for early diagnosis of HSCC are necessary. Unfortunately, appropriate biomarkers for clinical diagnosis and prognosis have not been identified yet. However, recent progresses in quantitative proteomics have offered opportunities to identify plasma proteins as biomarkers for HSCC. In the present study, plasma samples were analyzed by two-dimensional differential gel electrophoresis (2D-DIGE), and differentially expressed proteins were identified by matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS). A total of 26 proteins representing 12 unique gene products were identified. The up-regulation proteins were alpha-2-HS-glycoprotein (AHSG), complement C4-B, haptoglobin, C-reactive protein, and ceruloplasmin, whereas the down-regulation proteins were serum albumin, angiotensinogen, alpha-1-antichymotrypsin, Ig gamma-3 chain C region, fibrinogen gamma chain, apolipoprotein A-I, and Ig kappa chain C region. Among all the differentially expressed proteins, AHSG was validated by western blot and ELISA. The results were consistent with the data from 2D-DIGE, further suggesting that AHSG may be employed as a potential biomarker for the early diagnosis of HSCC. In summary, this study was the first to use 2D-DIGE and MALDI-TOF/TOF platform to identify the potential plasma biomarkers for HSCC. The plasma AHSG showed great potential for HSCC screening. PMID:26464644

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. Plasma chemistry in peregrine falcons (Falco peregrinus): Reference values and physiological variations of importance for interpretation.

    PubMed

    Lumeij, J T; Remple, J D; Remple, C J; Riddle, K E

    1998-01-01

    Reference values (inner limits of the percentiles P(2.5) and P(97.5) are given with a probability of 95%) for 21 plasma chemical variables were established in 79 peregrine falcons (Falco peregrinus). The following values were established: urea 0.8 to 3.9 mmol/l, creatinine 24 to 64 mumol/l, glucose 16.5 to 22.0 mmol/l, sodium 150 to 170 mmol/l, chloride 114 to 131 mmol/I, inorganic phosphorus 0.55 to 1.53 mmol/l, osmolal-ity 322 to 356 mOsmol/kg, alkaline phosphatase 31 to 121 IU/l, alanine aminotransferase 29 to 90 IU/l, aspartate aminotransferase 34 to 116 U/l, gamma glutamyl transferase 0 to 3 IU/l, lactate dehydrogenase 1008 to 2650 IU/l, creatine kinase 120 to 442 IU/l, cholinesterase 143 to 325 IU/1, glutamate dehydrogenase < 8 IU/l, total bile acids 5 to 69 mumol/l, uric acid 253 to 995 mumol/l, total protein 24 to 39 g/l, albumin 12.7 to 22.4 g/l. Reference values for the calculated albumin/globulin (A/G) ratio were 0.8 to 24. Based on previous studies, reference values for calcium were established using an adjustment formula using plasma total protein concentrations (before correction 1.86 to 2.49, after correction 1.97 to 2.46 mmol/l). Results of plasma potassium concentrations were erratic which was shown to be due to a time lag between sample collection and separation of plasma and cells.

  14. Feeding milk replacer instead of whole milk affects blood plasma proteome and lipid profile in preruminant calves.

    PubMed

    Lepczyński, A; Herosimczyk, A; Ożgo, M; Skrzypczak, W F

    2015-01-01

    The study was undertaken to determine the effect of feeding milk or milk-replacer on the blood plasma proteome and lipid profile in calves during the second week of life. Feeding milk-replacer significantly decreased the expression of plasma apoA-I. Age of calves affected apoA-I expression, which was higher on the 8th than on the 11th and 14th day of life. A significant effect of interaction between diet and age was also observed. The expression of apoA-IV, was significantly affected by diet and was lower in calves fed milk replacer. Expression of this protein was significantly lower at the 8th day of life and was up-regulated in the calves fed milk-replacer at the second week of life. Calves fed milk-replacer had greater expression of haptoglobin, which differed significantly between days of blood sampling, being higher on the 8th than on the 11th and 14th day. The interactive effect of diet and age affected haptoglobin expression, which was successively down-regulated in calves fed milk re- placer. Diet had a significant effect on the plasma lipid profile. Animals fed milk had a greater concentration of TC, HDLC and LDLC. The composition of milk-replacer, especially fat source, is probably the main factor that affects expression of proteins involved in cholesterol metabolism and level of components of lipid profile in calves fed formula. We claim that the initially increased level of haptoglobin, followed by its decrease during the second week of life in calves fed milk-replacer may indicate the presence of short-term stress induced by changes in the feeding system. PMID:25928915

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

    PubMed

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

    2007-01-01

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

  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. Quantitative plasma proteome analysis reveals aberrant level of blood coagulation-related proteins in nasopharyngeal carcinoma.

    PubMed

    Peng, Pei-Hua; Wu, Chih-Ching; Liu, Shu-Chen; Chang, Kai-Ping; Chen, Chi-De; Chang, Ya-Ting; Hsu, Chia-Wei; Chang, Yu-Sun; Yu, Jau-Song

    2011-05-01

    Nasopharyngeal carcinoma (NPC), one of the most common cancers in Southeast Asia, is not easily diagnosed until advanced stages. To discover potential biomarkers for improving NPC diagnosis, we herein identified the aberrant plasma proteins in NPC patients. We first removed the top-seven abundant proteins from plasma samples of healthy controls and NPC patients, and then labeled the samples with different fluorescent cyanine dyes. The labeled samples were then mixed equally and fractionated with ion-exchange chromatography followed by SDS-PAGE. Proteins showing altered levels in NPC patients were identified by in-gel tryptic digestion and LC-MS/MS. When the biological roles of the 45 identified proteins were assessed via MetaCore™ analysis, the blood coagulation pathway emerged as the most significantly altered pathway in NPC plasma. Plasma kallikrein (KLKB1) and thrombin-antithrombin III complex (TAT) were chosen for evaluation as the candidate NPC biomarkers because of their involvement in blood coagulation. ELISAs confirmed the elevation of their plasma levels in NPC patients versus healthy controls. Western blot and activity assays further showed that the KLKB1 active form was significantly increased in NPC plasma. Collectively, our results identified the significant alteration of blood coagulation pathway in NPC patients, and KLKB1 and TAT may represent the potential NPC biomarkers.

  18. 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. PMID:22275182

  19. 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.

  20. 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.

  1. 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

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

    PubMed

    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

  3. "Omics" of Selenium Biology: A Prospective Study of Plasma Proteome Network Before and After Selenized-Yeast Supplementation in Healthy Men.

    PubMed

    Sinha, Indu; Karagoz, Kubra; Fogle, Rachel L; Hollenbeak, Christopher S; Zea, Arnold H; Arga, Kazim Y; Stanley, Anne E; Hawkes, Wayne C; Sinha, Raghu

    2016-04-01

    Low selenium levels have been linked to a higher incidence of cancer and other diseases, including Keshan, Chagas, and Kashin-Beck, and insulin resistance. Additionally, muscle and cardiovascular disorders, immune dysfunction, cancer, neurological disorders, and endocrine function have been associated with mutations in genes encoding for selenoproteins. Selenium biology is complex, and a systems biology approach to study global metabolomics, genomics, and/or proteomics may provide important clues to examining selenium-responsive markers in circulation. In the current investigation, we applied a global proteomics approach on plasma samples collected from a previously conducted, double-blinded placebo controlled clinical study, where men were supplemented with selenized-yeast (Se-Yeast; 300 μg/day, 3.8 μmol/day) or placebo-yeast for 48 weeks. Proteomic analysis was performed by iTRAQ on 8 plasma samples from each arm at baseline and 48 weeks. A total of 161 plasma proteins were identified in both arms. Twenty-two proteins were significantly altered following Se-Yeast supplementation and thirteen proteins were significantly changed after placebo-yeast supplementation in healthy men. The differentially expressed proteins were involved in complement and coagulation pathways, immune functions, lipid metabolism, and insulin resistance. Reconstruction and analysis of protein-protein interaction network around selected proteins revealed several hub proteins. One of the interactions suggested by our analysis, PHLD-APOA4, which is involved in insulin resistance, was subsequently validated by Western blot analysis. Our systems approach illustrates a viable platform for investigating responsive proteomic profile in 'before and after' condition following Se-Yeast supplementation. The nature of proteins identified suggests that selenium may play an important role in complement and coagulation pathways, and insulin resistance. PMID:27027327

  4. 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.

  5. Plasma proteome profiles associated with diet-induced metabolic syndrome and the early onset of metabolic syndrome in a pig model.

    PubMed

    te Pas, Marinus F W; Koopmans, Sietse-Jan; Kruijt, Leo; Calus, Mario P L; Smits, Mari A

    2013-01-01

    Obesity and related diabetes are important health threatening multifactorial metabolic diseases and it has been suggested that 25% of all diabetic patients are unaware of their patho-physiological condition. Biomarkers for monitoring and control are available, but early stage predictive biomarkers enabling prevention of these diseases are still lacking. We used the pig as a model to study metabolic disease because humans and pigs share a multitude of metabolic similarities. Diabetes was chemically induced and control and diabetic pigs were either fed a high unsaturated fat (Mediterranean) diet or a high saturated fat/cholesterol/sugar (cafeteria) diet. Physiological parameters related to fat metabolism and diabetes were measured. Diabetic pigs' plasma proteome profiles differed more between the two diets than control pigs plasma proteome profiles. The expression levels of several proteins correlated well with (patho)physiological parameters related to the fat metabolism (cholesterol, VLDL, LDL, NEFA) and diabetes (Glucose) and to the diet fed to the animals. Studying only the control pigs as a model for metabolic syndrome when fed the two diets showed correlations to the same parameters but now more focused on insulin, glucose and abdominal fat depot parameters. We conclude that proteomic profiles can be used as a biomarker to identify pigs with developing metabolic syndrome (prediabetes) and diabetes when fed a cafeteria diet. It could be developed into a potential biomarkers for the early recognition of metabolic diseases.

  6. 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.

  7. Blood and Plasma Biochemistry Reference Intervals for Wild Juvenile American Alligators ( Alligator mississippiensis ).

    PubMed

    Hamilton, Matthew T; Kupar, Caitlin A; Kelley, Meghan D; Finger, John W; Tuberville, Tracey D

    2016-07-01

    : American alligators ( Alligator mississippiensis ) are one of the most studied crocodilian species in the world, yet blood and plasma biochemistry information is limited for juvenile alligators in their northern range, where individuals may be exposed to extreme abiotic and biotic stressors. We collected blood samples over a 2-yr period from 37 juvenile alligators in May, June, and July to establish reference intervals for 22 blood and plasma analytes. We observed no effect of either sex or blood collection time on any analyte investigated. However, our results indicate a significant correlation between a calculated body condition index and aspartate aminotransferase and creatine kinase. Glucose, total protein, and potassium varied significantly between sampling sessions. In addition, glucose and potassium were highly correlated between the two point-of-care devices used, although they were significantly lower with the i-STAT 1 CG8+ cartridge than with the Vetscan VS2 Avian/Reptile Rotor. The reference intervals presented herein should provide baseline data for evaluating wild juvenile alligators in the northern portion of their range. PMID:27224213

  8. 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, monoqtes 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.

  9. Numerical analysis of the non-equilibrium plasma flow in the gaseous electronics conference reference reactor

    NASA Astrophysics Data System (ADS)

    Bijie, Yang; Ning, Zhou; Quanhua, Sun

    2016-01-01

    The capacitively coupled plasma in the gaseous electronics conference reference reactor is numerically investigated for argon flow using a non-equilibrium plasma fluid model. The finite rate chemistry is adopted for the chemical non-equilibrium among species including neutral metastable, whereas a two-temperature model is employed to resolve the thermal non-equilibrium between electrons and heavy species. The predicted plasma density agrees very well with experimental data for the validation case. A strong thermal non-equilibrium is observed between heavy particles and electrons due to its low collision frequency, where the heavy species remains near ambient temperature for low pressure and low voltage conditions (0.1 Torr, 100 V). The effects of the operating parameters on the ion flux are also investigated, including the electrode voltage, chamber pressure, and gas flow rate. It is found that the ion flux can be increased by either elevating the electrode voltage or lowering the gas pressure. Project supported by the National Natural Science Foundation of China (Nos. 11372325, 11475239).

  10. 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

  11. 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. PMID:26780773

  12. 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.

  13. 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.

  14. Combination of virtual and experimental 2DE together with ESI LC-MS/MS gives a clearer view about proteomes of human cells and plasma.

    PubMed

    Naryzhny, Stanislav N; Zgoda, Victor G; Maynskova, Maria A; Novikova, Svetlana E; Ronzhina, Natalia L; Vakhrushev, Igor V; Khryapova, Elena V; Lisitsa, Andrey V; Tikhonova, Olga V; Ponomarenko, Elena A; Archakov, Alexander I

    2016-01-01

    Virtual and experimental 2DE coupled with ESI LC-MS/MS was introduced to obtain better representation of the information about human proteome. The proteins from HEPG2 cells and human blood plasma were run by 2DE. After staining and protein spot identification by MALDI-TOF MS, the protein maps were generated. The experimental physicochemical parameters (pI/Mw) of the proteoforms further detected by ESI LC-MS/MS in these spots were obtained. Next, the theoretical pI and Mw of identified proteins were calculated using program Compute pI/Mw (http://web.expasy.org/compute_pi/pi_tool-doc.html). Accordingly, the relationship between theoretical and experimental parameters was analyzed, and the correlation plots were built. Additionally, virtual/experimental information about different protein species/proteoforms from the same genes was extracted. As it was revealed from the plots, the major proteoforms detected in HepG2 cell line have pI/Mw parameters similar to theoretical values. In opposite, the minor protein species have mainly very different from theoretical pI and Mw parameters. A similar situation was observed in plasma in much higher degree. It means that minor protein species are heavily modified in cell and even more in plasma proteome.

  15. Proteomic Identification of Protein Targets for 15-Deoxy-Δ12,14-Prostaglandin J2 in Neuronal Plasma Membrane

    PubMed Central

    Yamamoto, Yasuhiro; Takase, Kenkichi; Kishino, Junji; Fujita, Megumi; Okamura, Noboru; Sakaeda, Toshiyuki; Fujimoto, Masafumi; Yagami, Tatsurou

    2011-01-01

    15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is one of factors contributed to the neurotoxicity of amyloid β (Aβ), a causative protein of Alzheimer's disease. Type 2 receptor for prostaglandin D2 (DP2) and peroxysome-proliferator activated receptorγ (PPARγ) are identified as the membrane receptor and the nuclear receptor for 15d-PGJ2, respectively. Previously, we reported that the cytotoxicity of 15d-PGJ2 was independent of DP2 and PPARγ, and suggested that 15d-PGJ2 induced apoptosis through the novel specific binding sites of 15d-PGJ2 different from DP2 and PPARγ. To relate the cytotoxicity of 15d-PGJ2 to amyloidoses, we performed binding assay [3H]15d-PGJ2 and specified targets for 15d-PGJ2 associated with cytotoxicity. In the various cell lines, there was a close correlation between the susceptibilities to 15d-PGJ2 and fibrillar Aβ. Specific binding sites of [3H]15d-PGJ2 were detected in rat cortical neurons and human bronchial smooth muscle cells. When the binding assay was performed in subcellular fractions of neurons, the specific binding sites of [3H]15d-PGJ2 were detected in plasma membrane, nuclear and cytosol, but not in microsome. A proteomic approach was used to identify protein targets for 15d-PGJ2 in the plasma membrane. By using biotinylated 15d-PGJ2, eleven proteins were identified as biotin-positive spots and classified into three different functional proteins: glycolytic enzymes (Enolase2, pyruvate kinase M1 (PKM1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)), molecular chaperones (heat shock protein 8 and T-complex protein 1 subunit α), cytoskeletal proteins (Actin β, F-actin-capping protein, Tubulin β and Internexin α). GAPDH, PKM1 and Tubulin β are Aβ-interacting proteins. Thus, the present study suggested that 15d-PGJ2 plays an important role in amyloidoses not only in the central nervous system but also in the peripheral tissues. PMID:21445266

  16. A proteomic study reveals the diversified distribution of plasma membrane-associated proteins in rat hepatocytes.

    PubMed

    Li, Xuanwen; Cao, Jia; Jin, Qihui; Xie, Chunliang; He, Quanyuan; Cao, Rui; Xiong, Jixian; Chen, Ping; Wang, Xianchun; Liang, Songping

    2008-06-01

    To investigate the heterogeneous protein composition of highly polarized hepatocyte plasma membrane (PM), three PM-associated subfractions were obtained from freshly isolated rat hepatocytes using density gradient centrifugation. The origins of the three subfractions were determined by morphological analysis and western blotting. The proteins were subjected to either one-dimensional (1-D) SDS-PAGE or two-dimensional (2-D) benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE before nano-Liquid Chromatography-Electrospray Ionization--tandem mass spectrometry analysis (LC-ESI-MS/MS). A total of 613 non-redundant proteins were identified, among which 371 (60.5%) proteins were classified as PM or membrane-associated proteins according to GO annotations and the literatures and 32.4% had transmembrane domains. PM proteins from microsomal portion possessed the highest percentage of transmembrane domain, about 46.5% of them containing at least one transmembrane domain. In addition to proteins known to be located at polarized liver PM regions, such as asialoglycoprotein receptor 2, desmoplakin and bile salt export pump, several proteins which had the potential to become novel subfraction-specific proteins were also identified, such as annexin a6, pannexin and radixin. Our analysis also evaluated the application of 1-D SDS-PAGE and 2-D 16-BAC/SDS-PAGE on the separation of integral membrane proteins.

  17. Inductively-coupled plasma mass spectrometry in proteomics, metabolomics and metallomics studies.

    PubMed

    Mounicou, Sandra; Szpunar, Joanna; Lobinski, Ryszard

    2010-01-01

    The potential of inductively-coupled plasma mass spectrometry (ICP-MS) and its complementarity to soft- ionization MS techniques are discussed in the context of the analysis for biomolecules. ICP-MS offers detection limits in the attomolar range, regardless of the molecular environment of the target element. The sensitivity is hardly affected by the sample matrix, chromatographic mobile phase, or co-eluted compounds. The abundance sensitivity over six decades and the linear dynamic range over nine decades make simultaneous multi-isotopic analysis routinely possible. The manuscript discusses the state-of-the-art of ICP-MS for the detection of proteins in gel electrophoresis and of peptides in 2D high-performance liquid chromatography. The possibilities of quantification to the degree of some post-translational modifications are highlighted. Attention is also paid to the role of ICP-MS in protein quantification via metal-coded labeling and to the use of differentially-labeled antibodies for the multiplexed biomarker analysis. The key role of ICP-MS in the emerging area of metallomics is briefly discussed.

  18. Proteomic analysis of mouse liver plasma membrane: use of differential extraction to enrich hydrophobic membrane proteins.

    PubMed

    Zhang, Lijun; Xie, Jinyun; Wang, Xi'e; Liu, Xiaohui; Tang, Xinke; Cao, Rui; Hu, Weijun; Nie, Song; Fan, Chunming; Liang, Songping

    2005-11-01

    To comprehensively identify proteins of liver plasma membrane (PM), we isolated PMs from mouse liver by sucrose density gradient centrifugation. An optimized extraction method for whole PM proteins and several methods of differential extraction expected to enrich hydrophobic membrane proteins were tested. The extracted PM proteins were separated by 2-DE, and were identified by MALDI-TOF-MS, and ESI-quadrupole-TOF MS. As the complementary method, 1-DE-MS/MS was also used to identify PM proteins. The optimized lysis buffer containing urea, thiourea, CHAPS and NP-40 was able to extract more PM proteins, and treatment of PM samples with chloroform/methanol and sodium carbonate led to enrichment of more hydrophobic PM proteins. From the mouse liver PM fraction, 175 non-redundant gene products were identified, of which 88 (about 50%) were integral membrane proteins with one to seven transmembrane domains. The remaining products were probably membrane-associated and cytosolic proteins. The function distribution of all the identified liver PM proteins was analyzed; 40% represented enzymes, 12% receptors and 9% proteins with unknown function.

  19. 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.

  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.

  1. Proteomic analysis of rat plasma with experimental autoimmune uveitis based on label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS).

    PubMed

    Guo, Dadong; Gu, Peiming; Liu, Zhengfeng; Tang, Kai; Du, Yuxiang; Bi, Hongsheng

    2015-01-22

    Uveitis is a severe autoimmune eye disease that can cause intraocular inflammation even lead to severe vision loss, and the occurrence of uveitis can be closely associated with abnormal expression of proteins. However, the abnormally expressed proteins involved in uveitis are not well identified. Using liquid chromatography-tandem mass spectrometry technique, we examined the alterations in proteomic expression profiling in rat plasma specimens related to experimental autoimmune uveitis (EAU) versus normal samples. In addition, the experimental verification was further performed using enzyme-linked immunosorbent assay (ELISA) for abnormally expressed proteins in EAU rat plasma. The results indicate that 62 proteins were upregulated and 106 proteins were downregulated in plasma from EAU rats compared with those in saline-treated samples. In the meantime, we observed that the plasma level of complement component 3 in EAU rats was upregulated versus saline-treated rats (from 92.32μg/mL to 168.92μg/mL), whereas the level of interleukin-1 receptor accessory protein was downregulated (from 1120.97pg/mL to 798.39pg/mL), and these results were highly in agreement with those of mass spectrometry determination. Taken together, our results indicate that liquid chromatography-tandem mass spectrometry analysis possesses a good resolution for peptides in plasma, and the findings will provide the baseline plasma dataset for EAU rats and the relevant information can contribute to future studies on the understanding the mechanism of uveitis.

  2. 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

  3. 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

  4. 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/.

  5. 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

  6. Reference study to characterize plasma and magnetic properties of ultracool atmospheres

    NASA Astrophysics Data System (ADS)

    Rodríguez-Barrera, M. I.; Helling, Ch.; Stark, C. R.; Rice, A. M.

    2015-12-01

    Radio and X-ray emission from brown dwarfs (BDs) suggest that an ionized gas and a magnetic field with a sufficient flux density must be present. We perform a reference study for late M-dwarfs (MD), BDs and giant gas planet to identify which ultracool objects are most susceptible to plasma and magnetic processes. Only thermal ionization is considered. We utilize the DRIFT-PHOENIX model grid where the local atmospheric structure is determined by the global parameters Teff, log (g) and [M/H]. Our results show that it is not unreasonable to expect Hα or radio emission to origin from BD atmospheres as in particular the rarefied upper parts of the atmospheres can be magnetically coupled despite having low degrees of thermal gas ionization. Such ultracool atmospheres could therefore drive auroral emission without the need for a companion's wind or an outgassing moon. The minimum threshold for the magnetic flux density required for electrons and ions to be magnetized is well above typical values of the global magnetic field of a BD and a giant gas planet. Na+, K+ and Ca+ are the dominating electron donors in low-density atmospheres (low log(g), solar metallicity) independent of Teff. Mg+ and Fe+ dominate the thermal ionization in the inner parts of MD atmospheres. Molecules remain unimportant for thermal ionization. Chemical processes (e.g. cloud formation) affecting the most abundant electron donors, Mg and Fe, will have a direct impact on the state of ionization in ultracool atmospheres.

  7. 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.

  8. 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.

  9. 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.

  10. 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. PMID:27293722

  11. 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

  12. 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).

  13. Blood-related proteomics.

    PubMed

    Liumbruno, Giancarlo; D'Alessandro, Angelo; Grazzini, Giuliano; Zolla, Lello

    2010-01-01

    Blood-related proteomics is an emerging field, recently gaining momentum. Indeed, a wealth of data is now available and a plethora of groups has contributed to add pieces to the jigsaw puzzle of protein complexity within plasma and blood cells. In this review article we purported to sail across the mare magnum of the actual knowledge in this research endeavour. The main strides in proteomic investigations on red blood cells, platelets, plasma and white blood cells are hereby presented in a chronological order. Moreover, a glance is given at prospective studies which promise to shift the focus of attention from the end product to its provider, the donor, in a sort of Kantian "Copernican revolution". A well-rounded portrait of the usefulness of proteomics in blood-related research is accurately given. In particular, proteomic tools could be adopted to follow the main steps of the blood-banking production processes (a comparison of collection methods, pathogen inactivation techniques, storage protocols). Thus proteomics has been recently transformed from a mere basic-research extremely-expensive toy into a dramatically-sensitive and efficient eye-lens to either delve into the depths of the molecular mechanisms of blood and blood components or to establish quality parameters in the blood-banking production chain totally anew. PMID:19567275

  14. Proteomics-based identification and validation of novel plasma biomarkers phospholipid transfer protein and mannan-binding lectin serine protease-1 in age-related macular degeneration.

    PubMed

    Kim, Hye-Jung; Ahn, Seong Joon; Woo, Se Joon; Hong, Hye Kyoung; Suh, Eui Jin; Ahn, Jeeyun; Park, Ji Hyun; Ryoo, Na-Kyung; Lee, Ji Eun; Kim, Ki Woong; Park, Kyu Hyung; Lee, Cheolju

    2016-01-01

    Age-related macular degeneration (AMD) is a major cause of severe, progressive visual loss among the elderly. There are currently no established serological markers for the diagnosis of AMD. In this study, we carried out a large-scale quantitative proteomics analysis to identify plasma proteins that could serve as potential AMD biomarkers. We found that the plasma levels of phospholipid transfer protein (PLTP) and mannan-binding lectin serine protease (MASP)-1 were increased in AMD patients relative to controls. The receiver operating characteristic curve based on data from an independent set of AMD patients and healthy controls had an area under the curve of 0.936 for PLTP and 0.716 for MASP-1, revealing excellent discrimination between the two groups. A proteogenomic combination model that incorporated PLTP and MASP-1 along with two known risk genotypes of age-related maculopathy susceptibility 2 and complement factor H genes further enhanced discriminatory power. Additionally, PLTP and MASP-1 mRNA and protein expression levels were upregulated in retinal pigment epithelial cells upon exposure to oxidative stress in vitro. These results indicate that PLTP and MASP-1 can serve as plasma biomarkers for the early diagnosis and treatment of AMD, which is critical for preventing AMD-related blindness. PMID:27605007

  15. Proteomics-based identification and validation of novel plasma biomarkers phospholipid transfer protein and mannan-binding lectin serine protease-1 in age-related macular degeneration

    PubMed Central

    Kim, Hye-Jung; Ahn, Seong Joon; Woo, Se Joon; Hong, Hye Kyoung; Suh, Eui Jin; Ahn, Jeeyun; Park, Ji Hyun; Ryoo, Na-Kyung; Lee, Ji Eun; Kim, Ki Woong; Park, Kyu Hyung; Lee, Cheolju

    2016-01-01

    Age-related macular degeneration (AMD) is a major cause of severe, progressive visual loss among the elderly. There are currently no established serological markers for the diagnosis of AMD. In this study, we carried out a large-scale quantitative proteomics analysis to identify plasma proteins that could serve as potential AMD biomarkers. We found that the plasma levels of phospholipid transfer protein (PLTP) and mannan-binding lectin serine protease (MASP)-1 were increased in AMD patients relative to controls. The receiver operating characteristic curve based on data from an independent set of AMD patients and healthy controls had an area under the curve of 0.936 for PLTP and 0.716 for MASP-1, revealing excellent discrimination between the two groups. A proteogenomic combination model that incorporated PLTP and MASP-1 along with two known risk genotypes of age-related maculopathy susceptibility 2 and complement factor H genes further enhanced discriminatory power. Additionally, PLTP and MASP-1 mRNA and protein expression levels were upregulated in retinal pigment epithelial cells upon exposure to oxidative stress in vitro. These results indicate that PLTP and MASP-1 can serve as plasma biomarkers for the early diagnosis and treatment of AMD, which is critical for preventing AMD-related blindness. PMID:27605007

  16. 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

  17. 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.

  18. 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.

  19. Identification of phosphorylated MYL12B as a potential plasma biomarker for septic acute kidney injury using a quantitative proteomic approach.

    PubMed

    Wu, Fan; Dong, Xiu-Juan; Li, Yan-Yan; Zhao, Yan; Xu, Qiu-Lin; Su, Lei

    2015-01-01

    Acute kidney injury (AKI) is a common and increasingly encountered complication in hospitalized patients with critical illness in intensive care units (ICU). According to the etiology, Sepsis-induced AKI (SAKI) is a leading contributor to AKI and significantly has very poor prognosis, which might be related to the late detection when the elevation of BUN and serum creatinine (SCr) is used. Many genes are up-regulated in the damaged kidney with the corresponding protein products appearing in plasma and urine. Some of these are candidate biomarkers for more timely diagnosis of SAKI. Therefore, extensive research efforts over this past decade have been directed at the discovery and validation of novel SAKI biomarkers to detect injury prior to changes in kidney function, a number of serum and urinary proteins, including NGAL, KIM-1, cystatin-C, IL-18, and L-FABP, have been identified for predicting SAKI before a rise in BUN and serum creatinine in several experimental and clinical trainings. Unfortunately, an ideal biomarker of SAKI with highly sensitivity and specificity has not been identified yet. Recent progresses in quantitative proteomics have offered opportunities to discover biomarkers for SAKI. In the present study, kidney tissue samples from SAKI mice were analyzed by two-dimensional differential gel electrophoresis (2D-DIGE), and 4 up-regulated proteins, which were actin (ACTB), myosin regulatory light chain 12B (MYL12B), myosin regulatory light polypeptide 9 (MYL9), and myosin regulatory light chain 12A (MYL12A) were identified by matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS). Among all the varied proteins, MYL12B was validated by western blot. Interestingly, there was no change between the SAKI and control kidney tissues, however, phosphorylated MYL12B was detected to be consistent with the proteomics data. Furthermore, phosphorylated MYL12B was found similarly to be increased in SAKI plasma

  20. Identification of phosphorylated MYL12B as a potential plasma biomarker for septic acute kidney injury using a quantitative proteomic approach

    PubMed Central

    Wu, Fan; Dong, Xiu-Juan; Li, Yan-Yan; Zhao, Yan; Xu, Qiu-Lin; Su, Lei

    2015-01-01

    Acute kidney injury (AKI) is a common and increasingly encountered complication in hospitalized patients with critical illness in intensive care units (ICU). According to the etiology, Sepsis-induced AKI (SAKI) is a leading contributor to AKI and significantly has very poor prognosis, which might be related to the late detection when the elevation of BUN and serum creatinine (SCr) is used. Many genes are up-regulated in the damaged kidney with the corresponding protein products appearing in plasma and urine. Some of these are candidate biomarkers for more timely diagnosis of SAKI. Therefore, extensive research efforts over this past decade have been directed at the discovery and validation of novel SAKI biomarkers to detect injury prior to changes in kidney function, a number of serum and urinary proteins, including NGAL, KIM-1, cystatin-C, IL-18, and L-FABP, have been identified for predicting SAKI before a rise in BUN and serum creatinine in several experimental and clinical trainings. Unfortunately, an ideal biomarker of SAKI with highly sensitivity and specificity has not been identified yet. Recent progresses in quantitative proteomics have offered opportunities to discover biomarkers for SAKI. In the present study, kidney tissue samples from SAKI mice were analyzed by two-dimensional differential gel electrophoresis (2D-DIGE), and 4 up-regulated proteins, which were actin (ACTB), myosin regulatory light chain 12B (MYL12B), myosin regulatory light polypeptide 9 (MYL9), and myosin regulatory light chain 12A (MYL12A) were identified by matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS). Among all the varied proteins, MYL12B was validated by western blot. Interestingly, there was no change between the SAKI and control kidney tissues, however, phosphorylated MYL12B was detected to be consistent with the proteomics data. Furthermore, phosphorylated MYL12B was found similarly to be increased in SAKI plasma

  1. Partial thromboplastin time test with kaolin: diagnosis of haemophilia and Christmas disease without natural reference plasmas.

    PubMed

    Knights, S F; Ingram, G I

    1967-07-01

    Deficiencies of factor VIII (in haemophilia) and factor IX (in Christmas disease) prolong the partial thromboplastin time. If normal plasma is treated with alumina, the factor VIII remains but the factor IX is removed and can subsequently be recovered by elution of the alumina. If a long partial thromboplastin time is found on investigating a male patient whose history suggests a life-long bleeding disorder, the plasma may be retested after adding either alumina-adsorbed normal plasma or eluate. If the patient's partial thromboplastin time is shortened (relative to the control) by adding adsorbed normal plasma the patient is likely to be a haemophiliac; but if it is shortened by adding eluate then he is likely to have Christmas disease. Practical details for carrying out these manoeuvres are given and experiments on the validity of the test described.

  2. The membrane proteome of the mouse lens fiber cell

    PubMed Central

    Wilmarth, Phillip A.; David, Larry L.

    2009-01-01

    Purpose Fiber cells of the ocular lens are bounded by a highly specialized plasma membrane. Despite the pivotal role that membrane proteins play in the physiology and pathophysiology of the lens, our knowledge of the structure and composition of the fiber cell plasma membrane remains fragmentary. In the current study, we utilized mass spectrometry-based shotgun proteomics to provide a comprehensive survey of the mouse lens fiber cell membrane proteome. Methods Membranes were purified from young mouse lenses and subjected to MudPIT (Multidimensional protein identification technology) analysis. The resulting proteomic data were analyzed further by reference to publically available microarray databases. Results More than 200 membrane proteins were identified by MudPIT, including Type I, Type II, Type III (multi-pass), lipid-anchored, and GPI-anchored membrane proteins, in addition to membrane-associated cytoskeletal elements and extracellular matrix components. The membrane proteins of highest apparent abundance included Mip, Lim2, and the lens-specific connexin proteins Gja3, Gja8, and Gje1. Significantly, many proteins previously unsuspected in the lens were also detected, including proteins with roles in cell adhesion, solute transport, and cell signaling. Conclusions The MudPIT technique constitutes a powerful technique for the analysis of the lens membrane proteome and provides valuable insights into the composition of the lens fiber cell unit membrane. PMID:19956408

  3. 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

  4. 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.

  5. 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).

  6. 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). PMID:20806654

  7. iTRAQ-based proteomic analysis of plasma reveals abnormalities in lipid metabolism proteins in chronic kidney disease-related atherosclerosis

    NASA Astrophysics Data System (ADS)

    Luczak, Magdalena; Formanowicz, Dorota; Marczak, Łukasz; Suszyńska-Zajczyk, Joanna; Pawliczak, Elżbieta; Wanic-Kossowska, Maria; Stobiecki, Maciej

    2016-09-01

    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.

  8. 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

  9. iTRAQ-based proteomic analysis of plasma reveals abnormalities in lipid metabolism proteins in chronic kidney disease-related atherosclerosis.

    PubMed

    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

  10. Quantitative Determination of Luminal and Abluminal Membrane Distributions of Transporters in Porcine Brain Capillaries by Plasma Membrane Fractionation and Quantitative Targeted Proteomics.

    PubMed

    Kubo, Yoshiyuki; Ohtsuki, Sumio; Uchida, Yasuo; Terasaki, Tetsuya

    2015-09-01

    Abluminal or luminal localization of transporter in plasma membranes at the blood-brain barrier (BBB) is critical for their physiological and pharmacological roles. Therefore, the purpose of this study was to develop a new method to investigate membrane localization of transporters, through quantitative measurement of protein expression levels in fractionated plasma membrane prepared from porcine brain capillaries. Luminal-abluminal distribution ratios were calculated from the results of quantitative targeted absolute proteomics of fractionated membranes, after correction for cross-contamination based on measurements of luminal and abluminal membrane markers. BCRP expression was greater at the luminal membrane than at the abluminal membrane, supporting the usefulness of the distribution ratio as a quantitative indicator of localization. The distribution ratios suggested luminal-dominant localizations of GLUT1 and OATP3A1, and abluminal-dominant localizations of ABCA1 and FATP1. For OATP3A1, ABCA1 and FATP1, these results require reconsideration of their functions at the BBB. Species differences were examined using expression levels normalized to Na(+) /K(+) -ATPase. BCRP expression is dominant over multidrug resistance 1 expression in porcine BBB, as in other primates including humans. This methodology for quantitative measurement of protein localization is expected to improve our understanding of the roles of transporters at the BBB, and should be applicable to other polarized cells.

  11. Diet-related reference values for plasma amino acids in newborns measured by reversed-phase HPLC.

    PubMed

    Scott, P H; Sandham, S; Balmer, S E; Wharton, B A

    1990-11-01

    We have measured by reversed-phase HPLC concentrations of amino acids in plasma in groups of 80 normal appropriate-weight term babies fed from birth either a casein formula (WhiteCap SMA, n = 26), a whey formula (Gold Cap SMA, n = 26), or breast milk (n = 28). They were studied from day 11 to week 15 postpartum. The trend was towards an increase in amino acid concentrations in plasma with age, more marked in formula-fed than in breast-fed infants. Reference values were derived for each group. Both formula-fed groups showed several differences from the breast-fed group. Detailed examination indicated that tyrosine, phenylalanine, and methionine concentrations were increased in the casein-fed group greater than 20% of the time, but only threonine was similarly increased in the whey-fed group. Other amino acids, different ones for each formula group, were increased less frequently. There were no consistent correlations with any aspect of infant growth. Appropriate reference values are important for interpreting amino acid concentrations in plasma from newborns and for evaluating the effects of any future dietary modifications to infant formulas. HPLC analysis provides a suitable highly sensitive method for undertaking such studies.

  12. Molecular Biologist's Guide to Proteomics

    PubMed Central

    Graves, Paul R.; Haystead, Timothy A. J.

    2002-01-01

    The emergence of proteomics, the large-scale analysis of proteins, has been inspired by the realization that the final product of a gene is inherently more complex and closer to function than the gene itself. Shortfalls in the ability of bioinformatics to predict both the existence and function of genes have also illustrated the need for protein analysis. Moreover, only through the study of proteins can posttranslational modifications be determined, which can profoundly affect protein function. Proteomics has been enabled by the accumulation of both DNA and protein sequence databases, improvements in mass spectrometry, and the development of computer algorithms for database searching. In this review, we describe why proteomics is important, how it is conducted, and how it can be applied to complement other existing technologies. We conclude that currently, the most practical application of proteomics is the analysis of target proteins as opposed to entire proteomes. This type of proteomics, referred to as functional proteomics, is always driven by a specific biological question. In this way, protein identification and characterization has a meaningful outcome. We discuss some of the advantages of a functional proteomics approach and provide examples of how different methodologies can be utilized to address a wide variety of biological problems. PMID:11875127

  13. 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

  14. 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.

  15. 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. PMID:27468020

  16. 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

  17. 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. PMID:21563312

  18. Proteomic profiling of naïve multiple myeloma patient plasma cells identifies pathways associated with favourable response to bortezomib-based treatment regimens.

    PubMed

    Dytfeld, Dominik; Rosebeck, Shaun; Kandarpa, Malathi; Mayampurath, Anoop; Mellacheruvu, Dattatreya; Alonge, Mattina M; Ngoka, Lambert; Jasielec, Jagoda; Richardson, Paul G; Volchenboum, Samuel; Nesvizhskii, Alexey I; Sreekumar, Arun; Jakubowiak, Andrzej J

    2015-07-01

    Toward our goal of personalized medicine, we comprehensively profiled pre-treatment malignant plasma cells from multiple myeloma patients and prospectively identified pathways predictive of favourable response to bortezomib-based treatment regimens. We utilized two complementary quantitative proteomics platforms to identify differentially-regulated proteins indicative of at least a very good partial response (VGPR) or complete response/near complete response (CR/nCR) to two treatment regimens containing either bortezomib, liposomal doxorubicin and dexamethasone (VDD), or lenalidomide, bortezomib and dexamethasone (RVD). Our results suggest enrichment of 'universal response' pathways that are common to both treatment regimens and are probable predictors of favourable response to bortezomib, including a subset of endoplasmic reticulum stress pathways. The data also implicate pathways unique to each regimen that may predict sensitivity to DNA-damaging agents, such as mitochondrial dysfunction, and immunomodulatory drugs, which was associated with acute phase response signalling. Overall, we identified patterns of tumour characteristics that may predict response to bortezomib-based regimens and their components. These results provide a rationale for further evaluation of the protein profiles identified herein for targeted selection of anti-myeloma therapy to increase the likelihood of improved treatment outcome of patients with newly-diagnosed myeloma. PMID:25824111

  19. A Shotgun Proteomic Approach Reveals That Fe Deficiency Causes Marked Changes in the Protein Profiles of Plasma Membrane and Detergent-Resistant Microdomain Preparations from Beta vulgaris Roots.

    PubMed

    Gutierrez-Carbonell, Elain; Takahashi, Daisuke; Lüthje, Sabine; González-Reyes, José Antonio; Mongrand, Sébastien; Contreras-Moreira, Bruno; Abadía, Anunciación; Uemura, Matsuo; Abadía, Javier; López-Millán, Ana Flor

    2016-08-01

    In the present study we have used label-free shotgun proteomic analysis to examine the effects of Fe deficiency on the protein profiles of highly pure sugar beet root plasma membrane (PM) preparations and detergent-resistant membranes (DRMs), the latter as an approach to study microdomains. Altogether, 545 proteins were detected, with 52 and 68 of them changing significantly with Fe deficiency in PM and DRM, respectively. Functional categorization of these proteins showed that signaling and general and vesicle-related transport accounted for approximately 50% of the differences in both PM and DRM, indicating that from a qualitative point of view changes induced by Fe deficiency are similar in both preparations. Results indicate that Fe deficiency has an impact in phosphorylation processes at the PM level and highlight the involvement of signaling proteins, especially those from the 14-3-3 family. Lipid profiling revealed Fe-deficiency-induced decreases in phosphatidic acid derivatives, which may impair vesicle formation, in agreement with the decreases measured in proteins related to intracellular trafficking and secretion. The modifications induced by Fe deficiency in the relative enrichment of proteins in DRMs revealed the existence of a group of cytoplasmic proteins that appears to be more attached to the PM in conditions of Fe deficiency. PMID:27321140

  20. A Shotgun Proteomic Approach Reveals That Fe Deficiency Causes Marked Changes in the Protein Profiles of Plasma Membrane and Detergent-Resistant Microdomain Preparations from Beta vulgaris Roots.

    PubMed

    Gutierrez-Carbonell, Elain; Takahashi, Daisuke; Lüthje, Sabine; González-Reyes, José Antonio; Mongrand, Sébastien; Contreras-Moreira, Bruno; Abadía, Anunciación; Uemura, Matsuo; Abadía, Javier; López-Millán, Ana Flor

    2016-08-01

    In the present study we have used label-free shotgun proteomic analysis to examine the effects of Fe deficiency on the protein profiles of highly pure sugar beet root plasma membrane (PM) preparations and detergent-resistant membranes (DRMs), the latter as an approach to study microdomains. Altogether, 545 proteins were detected, with 52 and 68 of them changing significantly with Fe deficiency in PM and DRM, respectively. Functional categorization of these proteins showed that signaling and general and vesicle-related transport accounted for approximately 50% of the differences in both PM and DRM, indicating that from a qualitative point of view changes induced by Fe deficiency are similar in both preparations. Results indicate that Fe deficiency has an impact in phosphorylation processes at the PM level and highlight the involvement of signaling proteins, especially those from the 14-3-3 family. Lipid profiling revealed Fe-deficiency-induced decreases in phosphatidic acid derivatives, which may impair vesicle formation, in agreement with the decreases measured in proteins related to intracellular trafficking and secretion. The modifications induced by Fe deficiency in the relative enrichment of proteins in DRMs revealed the existence of a group of cytoplasmic proteins that appears to be more attached to the PM in conditions of Fe deficiency.

  1. Proteomic profiling of naïve multiple myeloma patient plasma cells identifies pathways associated with favourable response to bortezomib-based treatment regimens.

    PubMed

    Dytfeld, Dominik; Rosebeck, Shaun; Kandarpa, Malathi; Mayampurath, Anoop; Mellacheruvu, Dattatreya; Alonge, Mattina M; Ngoka, Lambert; Jasielec, Jagoda; Richardson, Paul G; Volchenboum, Samuel; Nesvizhskii, Alexey I; Sreekumar, Arun; Jakubowiak, Andrzej J

    2015-07-01

    Toward our goal of personalized medicine, we comprehensively profiled pre-treatment malignant plasma cells from multiple myeloma patients and prospectively identified pathways predictive of favourable response to bortezomib-based treatment regimens. We utilized two complementary quantitative proteomics platforms to identify differentially-regulated proteins indicative of at least a very good partial response (VGPR) or complete response/near complete response (CR/nCR) to two treatment regimens containing either bortezomib, liposomal doxorubicin and dexamethasone (VDD), or lenalidomide, bortezomib and dexamethasone (RVD). Our results suggest enrichment of 'universal response' pathways that are common to both treatment regimens and are probable predictors of favourable response to bortezomib, including a subset of endoplasmic reticulum stress pathways. The data also implicate pathways unique to each regimen that may predict sensitivity to DNA-damaging agents, such as mitochondrial dysfunction, and immunomodulatory drugs, which was associated with acute phase response signalling. Overall, we identified patterns of tumour characteristics that may predict response to bortezomib-based regimens and their components. These results provide a rationale for further evaluation of the protein profiles identified herein for targeted selection of anti-myeloma therapy to increase the likelihood of improved treatment outcome of patients with newly-diagnosed myeloma.

  2. Quantitative Proteomics Reveals that Plasma Membrane Microdomains From Poplar Cell Suspension Cultures Are Enriched in Markers of Signal Transduction, Molecular Transport, and Callose Biosynthesis*

    PubMed Central

    Srivastava, Vaibhav; Malm, Erik; Sundqvist, Gustav; Bulone, Vincent

    2013-01-01

    The plasma membrane (PM) is a highly dynamic interface that contains detergent-resistant microdomains (DRMs). The aim of this work was to determine the main functions of such microdomains in poplar through a proteomic analysis using gel-based and solution (iTRAQ) approaches. A total of 80 proteins from a limited number of functional classes were found to be significantly enriched in DRM relative to PM. The enriched proteins are markers of signal transduction, molecular transport at the PM, or cell wall biosynthesis. Their intrinsic properties are presented and discussed together with the biological significance of their enrichment in DRM. Of particular importance is the significant and specific enrichment of several callose [(1→3)-β-glucan] synthase isoforms, whose catalytic activity represents a final response to stress, leading to the deposition of callose plugs at the surface of the PM. An integrated functional model that connects all DRM-enriched proteins identified is proposed. This report is the only quantitative analysis available to date of the protein composition of membrane microdomains from a tree species. PMID:24051156

  3. 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. PMID:26811355

  4. Microsomal proteomics.

    PubMed

    Wong, Diana M; Adeli, Khosrow

    2009-01-01

    Proteomic profiling of subcellular compartments has many advantages over traditional proteomic approaches using whole cell lysates as it allows for detailed proteome analysis of a specific organelle and corresponding functional characteristics. The microsome is a critical, membranous compartment involved in the synthesis, sorting, and secretion of proteins as well as other metabolic functions. This chapter will describe detailed methods for the isolation of microsomal organelles including the ER, Golgi, and prechylomicron transport vesicle (PCTV), a recently identified vesicular system involved in intestinal lipoprotein assembly and secretion. Particular focus is given to the isolation of microsomes from primary hepatocytes and enterocytes freshly isolated from rodent liver and intestinal tissue, and their proteomic profiling using a combination of two-dimensional gel electrophoresis and mass spectrometry.

  5. 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.

  6. 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.

  7. Proteomics of MUC1-containing lipid rafts from plasma membranes and exosomes of human breast carcinoma cells MCF-7.

    PubMed

    Staubach, Simon; Razawi, Hanieh; Hanisch, Franz-Georg

    2009-05-01

    Apically expressed human MUC1 is known to become endocytosed and either to re-enter the secretory pathway for recycling to the plasma membrane or to be exported by the cells via the formation of multi-vesicular bodies and the release of exosomes. By using recombinant fusion-tagged MUC1 as a bait protein we followed an anti-myc affinity-based approach for isolating subpopulations of lipid rafts from the plasma membranes and exosomes of MCF-7 breast cancer cells. MUC1(+) lipid rafts were not only found to contain genuine raft proteins (flotillin-1, prohibitin, G protein, annexin A2), but also raft-associated proteins linking these to the cytoskeleton (ezrin/villin-2, profilin II, HSP27, gamma-actin, beta-actin) or proteins in complexes with raft proteins, including the bait protein (HSP60, HSP70). Major overlaps were revealed for the subproteomes of plasma membranous and exosomal lipid raft preparations, indicating that MUC1 is sorted into subpopulations of rafts for its trafficking via flotillin-dependent pathways and export via exosomes.

  8. Aspartate aminotransferase and alanine aminotransferase activities in plasma: statistical distributions, individual variations, and reference values.

    PubMed

    Siest, G; Schiele, F; Galteau, M M; Panek, E; Steinmetz, J; Fagnani, F; Gueguen, R

    1975-07-01

    The determination of frequency value (percentile limits) and the classification of the different variation factors allow us to define more and more homogeneous subpopulations as we use these factors for sorting. Using as our study population those persons coming to the Centre for Preventive Medicine, we were able to: (a) Describe and measure the significance and importance of physiological variations or of variations attributed to age--the latter largely related only to excessive weight, which it seems to us is often the case. (b) Establish a classification for variation factors; the recapitulatory table should be useful to clinical chemists in helping physicians interpret a laboratory test result that falls within the zone of incertitude. (c) Suggest a preliminary group of reference values for healthy subjects, to be used in interpreting a laboratory test in this way.

  9. 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.

  10. 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

  11. 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

  12. A proteomic study reveals novel insights into the diversity of aquaporin forms expressed in the plasma membrane of plant roots.

    PubMed Central

    Santoni, Véronique; Vinh, Joëlle; Pflieger, Delphine; Sommerer, Nicolas; Maurel, Christophe

    2003-01-01

    Aquaporins are channel proteins that facilitate the diffusion of water across cell membranes. The genome of Arabidopsis thaliana encodes 35 full-length aquaporin homologues. Thirteen of them belong to the plasma membrane intrinsic protein (PIP) subfamily and predominantly sit at the plasma membrane (PM). In the present work we combine separations of membrane proteins (by one- and two-dimensional gel electrophoresis) with identification by MS (matrix-assisted laser-desorption ionization-time-of-flight and electrospray-ionization tandem MS) to take an inventory of aquaporin isoforms expressed in the PM of Arabidopsis thaliana roots. Our analysis provides direct evidence for the expression of five PIPs (PIP1;1, PIP1;5, PIP2;1, PIP2;2 and PIP2;7) in the root PM and suggests the presence of at least three other PIP isoforms. In addition, we show that the same PIP isoform can be present under several forms with distinct isoelectric points. More specifically, we identify phosphorylated aquaporins in the PIP1 and PIP2 subgroups and suggest the existence of other post-translational modifications. Their identification should provide clues to reveal novel molecular mechanisms for aquaporin regulation. PMID:12678916

  13. 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.

  14. Two-dimensional proteome reference map of Prototheca zopfii revealed reduced metabolism and enhanced signal transduction as adaptation to an infectious life style.

    PubMed

    Murugaiyan, Jayaseelan; Weise, Christoph; von Bergen, Martin; Roesler, Uwe

    2013-09-01

    Biochemical, serological, and genetic analyses have identified two genotypes of Prototheca zopfii, a unicellular microalga belonging to the family Chlorellaceae. The P. zopfii genotype 1, abundantly present in cow barns and environment, remains nonpathogenic, while P. zopfii genotype 2 has been isolated from cows with bovine mastitis. The present study was carried out to identify the protein expression level difference between the pathogenic and nonpathogenic strains of P. zopfii. A total of 782 protein spots were observed on the 2D fluorescence difference gel electrophoresis (2D DIGE) gels among which 63 and 44 proteins were identified to be overexpressed in genotypes 1 and 2, respectively. The limited number of protein entries specific for Prototheca in public repositories resulted mainly in the identification of proteins described in other algae, microorganisms, or plants. Gene ontology (GO) analysis indicated reduced carbohydrate metabolism in genotype 1, while genotype 2 displayed enhanced DNA binding, kinase activity, and signal transduction. These effects point to metabolic and signaling adaptations in the pathogenic strain and provide insights into the evolution of otherwise highly similar strains. All MS data have been deposited in the ProteomeXchange with identifier PXD000126. PMID:23852777

  15. Comparative proteomics of root plasma membrane proteins reveals the involvement of calcium signalling in NaCl-facilitated nitrate uptake in Salicornia europaea

    PubMed Central

    Nie, Lingling; Feng, Juanjuan; Fan, Pengxiang; Chen, Xianyang; Guo, Jie; Lv, Sulian; Bao, Hexigeduleng; Jia, Weitao; Tai, Fang; Jiang, Ping; Wang, Jinhui; Li, Yinxin

    2015-01-01

    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 Ca2+ concentration ([Ca2+]cyt) was significantly elevated in S. europaea under NaCl treatment. The application of the Ca2+ 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 Ca2+ signalling was proposed. PMID:25956883

  16. 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

  17. 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

    2010-04-01

    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.

  18. DEVELOPMENT OF REFERENCE RANGES FOR PLASMA TOTAL CHOLINESTERASE AND BRAIN ACETYLCHOLINESTERASE ACTIVITY IN FREE-RANGING CARNABY'S BLACK-COCKATOOS (CALYPTORHYNCHUS LATIROSTRIS).

    PubMed

    Vaughan-Higgins, Rebecca; Vitali, Simone; Reiss, Andrea; Besier, Shane; Hollingsworth, Tom; Smith, Gerard

    2016-07-01

    Published avian reference ranges for plasma cholinesterase (ChE) and brain acetylcholinesterase (AChE) are numerous. However, a consistently reported recommendation is the need for species- and laboratory-specific reference ranges because of variables, including assay methods, sample storage conditions, season, and bird sex, age, and physiologic status. We developed normal reference ranges for brain AChE and plasma total ChE (tChE) activity for Carnaby's Black-Cockatoos (Calyptorhynchus latirostris) using a standardized protocol (substrate acetylthiocholine at 25 C). We report reference ranges for brain AChE (19-41 μmol/min per g, mean 21±6.38) and plasma tChE (0.41-0.53 μmol/min per mL, mean 0.47±0.11) (n=15). This information will be of use in the ongoing field investigation of a paresis-paralysis syndrome in the endangered Carnaby's Black-Cockatoos, suspected to be associated with exposure to anticholinesterase compounds and add to the paucity of reference ranges for plasma tChE and brain AChE in Australian psittacine birds.

  19. Age- and sex-related reference ranges for eight plasma constituents derived from randomly selected adults in a Scottish new town.

    PubMed Central

    Gardner, M D; Scott, R

    1980-01-01

    The results of analysis of blood specimens from randomly selected adults aged 19-88 years in the new town of Cumbernauld were used to establish age- and sex-related reference ranges by the centile method (central 95%) for plasma calcium, phosphate, total protein, albumin, globulins, urea, creatinine, and urate. The possible existence of a subpopulation with a higher reference range for urea is mooted. PMID:7400337

  20. Platelet proteomics.

    PubMed

    Zufferey, Anne; Fontana, Pierre; Reny, Jean-Luc; Nolli, Severine; Sanchez, Jean-Charles

    2012-01-01

    Platelets are small cell fragments, produced by megakaryocytes, in the bone marrow. They play an important role in hemostasis and diverse thrombotic disorders. They are therefore primary targets of antithrombotic therapies. They are implicated in several pathophysiological pathways, such as inflammation or wound repair. In blood circulation, platelets are activated by several pathways including subendothelial matrix and thrombin, triggering the formation of the platelet plug. Studying their proteome is a powerful approach to understand their biology and function. However, particular attention must be paid to different experimental parameters, such as platelet quality and purity. Several technologies are involved during the platelet proteome processing, yielding information on protein identification, characterization, localization, and quantification. Recent technical improvements in proteomics combined with inter-disciplinary strategies, such as metabolomic, transcriptomics, and bioinformatics, will help to understand platelets biological mechanisms. Therefore, a comprehensive analysis of the platelet proteome under different environmental conditions may contribute to elucidate complex processes relevant to platelet function regarding bleeding disorders or platelet hyperreactivity and identify new targets for antiplatelet therapy.

  1. The proteome of schizophrenia

    PubMed Central

    Nascimento, Juliana M; Martins-de-Souza, Daniel

    2015-01-01

    On observing schizophrenia from a clinical point of view up to its molecular basis, one may conclude that this is likely to be one of the most complex human disorders to be characterized in all aspects. Such complexity is the reflex of an intricate combination of genetic and environmental components that influence brain functions since pre-natal neurodevelopment, passing by brain maturation, up to the onset of disease and disease establishment. The perfect function of tissues, organs, systems, and finally the organism depends heavily on the proper functioning of cells. Several lines of evidence, including genetics, genomics, transcriptomics, neuropathology, and pharmacology, have supported the idea that dysfunctional cells are causative to schizophrenia. Together with the above-mentioned techniques, proteomics have been contributing to understanding the biochemical basis of schizophrenia at the cellular and tissue level through the identification of differentially expressed proteins and consequently their biochemical pathways, mostly in the brain tissue but also in other cells. In addition, mass spectrometry-based proteomics have identified and precisely quantified proteins that may serve as biomarker candidates to prognosis, diagnosis, and medication monitoring in peripheral tissue. Here, we review all data produced by proteomic investigation in the last 5 years using tissue and/or cells from schizophrenic patients, focusing on postmortem brain tissue and peripheral blood serum and plasma. This information has provided integrated pictures of the biochemical systems involved in the pathobiology, and has suggested potential biomarkers, and warrant potential targets to alternative treatment therapies to schizophrenia. PMID:27336025

  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 Cancer Proteomics?

    MedlinePlus

    ... gov The National Institutes of Health Clinical Proteomics Technologies for Cancer Contact Us Intranet Sign Up for ... of proteomics that involves the application of proteomic technologies on clinical specimens such as blood. Cancer, in ...

  4. Studies of plasma zinc, copper, caeruloplasmin, and growth hormone: with special reference to carcinoma of the bronchus.

    PubMed

    Andrews, G S

    1979-04-01

    The levels of plasma zinc, copper, caeruloplasmin, and growth hormone were determined in a group of normal people and in four groups of patients who were suffering from carcinoma of the bronchus, other forms of malignancy, chest illnesses, and diseases other than chest illness or malignancy. The plasma zinc was higher, and the plasma copper lower, in people without malignancy below the age of 30 years than they were in other age groups.It was confirmed that about 66% of patients with carcinoma of the bronchus had plasma zinc levels less than 11.5 mumol/l but low levels were also found in 23% of other cases of malignancy and in 9% of the other patients. In carcinoma of the bronchus the low plasma zinc was found to be associated with epidermoid and anaplastic tumours and was to some extent related to the duration of the disease. In carcinoma of the bronchus the plasma copper was found to be higher than in all other groups, and values higher than 26.5 mumol/l were considered to support a diagnosis of carcinoma of the bronchus. There was, however, no relationship between the increase in the plasma copper and the decrease in the plasma zinc.Raised caeruloplasmin levels above 420 mg/l were found in 65% of cases of carcinoma of the bronchus, and these high levels were usually associated with raised plasma copper. Growth hormone was normal in all groups except six patients with carcinoma of the bronchus with secondary carcinoma of the liver, in whom it was raised. Surgical operations lowered plasma zinc and raised growth hormone but did not affect plasma copper.A plasma zinc below 11.5 mumol/l is helpful in the diagnosis of carcinoma of the bronchus, but by itself it is not sufficiently specific to be considered diagnostic or to form a reliable screening test. A raised plasma copper and a raised plasma caeruloplasmin were useful supportive findings.

  5. 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.

  6. European and international collaboration in affinity proteomics.

    PubMed

    Stoevesandt, Oda; Taussig, Michael J

    2012-06-15

    In affinity proteomics, specific protein-binding molecules (a.k.a. binders), principally antibodies, are applied as reagents in proteome analysis. In recent years, advances in binder technologies have created the potential for an unprecedented view on protein expression and distribution patterns in plasma, cells and tissues and increasingly on protein function. Particular strengths of affinity proteomics methods include detecting proteins in their natural environments of cell or tissue, high sensitivity and selectivity for detection of low abundance proteins and exploiting binding actions such as functional interference in living cells. To maximise the use and impact of affinity reagents, it will be essential to create comprehensive, standardised binder collections. With this in mind, the EU FP7 programme AFFINOMICS (http://www.affinomics.org), together with the preceding EU programmes ProteomeBinders and AffinityProteome, aims to extend affinity proteomics research by generating a large-scale resource of validated protein-binding molecules for characterisation of the human proteome. Activity is directed at producing binders to about 1000 protein targets, primarily in signal transduction and cancer, by establishing a high throughput, coordinated production pipeline. An important aspect of AFFINOMICS is the development of highly efficient recombinant selection methods, based on phage, cell and ribosome display, capable of producing high quality binders at greater throughput and lower cost than hitherto. The programme also involves development of innovative and sensitive technologies for specific detection of target proteins and their interactions, and deployment of binders in proteomics studies of clinical relevance. The need for such binder generation programmes is now recognised internationally, with parallel initiatives in the USA for cancer (NCI) and transcription factors (NIH) and within the Human Proteome Organisation (HUPO). The papers in this volume of New

  7. Proteomic Investigations into Hemodialysis Therapy.

    PubMed

    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

  8. 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.

  9. Accounting for population variation in targeted proteomics

    SciTech Connect

    Fujimoto, Grant M.; Monroe, Matthew E.; Rodriguez, Larissa M.; Wu, Chaochao; MacLean, Brendan; Smith, Richard D.; MacCoss, Michael; 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.

  10. 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.

  11. Plant secretome proteomics

    PubMed Central

    Alexandersson, Erik; Ali, Ashfaq; Resjö, Svante; Andreasson, Erik

    2013-01-01

    The plant secretome refers to the set of proteins secreted out of the plant cell into the surrounding extracellular space commonly referred to as the apoplast. Secreted proteins maintain cell structure and acts in signaling and are crucial for stress responses where they can interact with pathogen effectors and control the extracellular environment. Typically, secreted proteins contain an N-terminal signal peptide and are directed through the endoplasmic reticulum/Golgi pathway. However, in plants many proteins found in the secretome lack such a signature and might follow alternative ways of secretion. This review covers techniques to isolate plant secretomes and how to identify and quantify their constituent proteins. Furthermore, bioinformatical tools to predict secretion signals and define the putative secretome are presented. Findings from proteomic studies and important protein families of plant secretomes, such as proteases and hydrolases, are highlighted. PMID:23378846

  12. 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

  13. 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

    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

  14. 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. PMID:21877445

  15. 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.

  16. Lipolytic proteomics.

    PubMed

    Schittmayer, Matthias; Birner-Gruenberger, Ruth

    2012-01-01

    Activity-based proteomics (ABP) employs small molecular probes to specifically label sets of enzymes based on their shared catalytic mechanism. Given that the vast majority of lipases belong to the family of serine hydrolases and share a nucleophilic active-site serine as part of a catalytic triad, activity-based probes are ideal tools to study lipases and lipolysis. Moreover, the ability of ABP to highlight or isolate specific subproteomes results in a massive decrease of sample complexity. Thereby, in-depth analysis of enzymes of interest with mass spectrometry becomes feasible. In this review, we cover probe design, technological developments, and applications of ABP of lipases, as well as give an overview of relevant identified proteins.

  17. 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.

  18. Legume proteomics: Progress, prospects, and challenges.

    PubMed

    Rathi, Divya; Gayen, Dipak; Gayali, Saurabh; Chakraborty, Subhra; Chakraborty, Niranjan

    2016-01-01

    Legumes are the major sources of food and fodder with strong commercial relevance, and are essential components of agricultural ecosystems owing to their ability to carry out endosymbiotic nitrogen fixation. In recent years, legumes have become one of the major choices of plant research. The legume proteomics is currently represented by more than 100 reference maps and an equal number of stress-responsive proteomes. Among the 48 legumes in the protein databases, most proteomic studies have been accomplished in two model legumes, soybean, and barrel medic. This review highlights recent contributions in the field of legume proteomics to comprehend the defence and regulatory mechanisms during development and adaptation to climatic changes. Here, we attempted to provide a concise overview of the progress in legume proteomics and discuss future developments in three broad perspectives: (i) proteome of organs/tissues; (ii) subcellular compartments; and (iii) spatiotemporal changes in response to stress. Such data mining may aid in discovering potential biomarkers for plant growth, in general, apart from essential components involved in stress tolerance. The prospect of integrating proteome data with genome information from legumes will provide exciting opportunities for plant biologists to achieve long-term goals of crop improvement and sustainable agriculture.

  19. Functional proteomics of synaptic plasma membrane ATP-ases of rat hippocampus: effect of l-acetylcarnitine and relationships with Dementia and Depression pathophysiology.

    PubMed

    Ferrari, Federica; Gorini, Antonella; Villa, Roberto Federico

    2015-06-01

    Synaptic energy state and mitochondrial dysfunction are crucial factors in many brain pathologies. l-acetylcarnitine, a natural derivative of carnitine, improves brain energy metabolism, and has been proposed for the Therapy of many neurological and psychiatric diseases. The effects of the drug on the maximum rate (Vmax) of enzymatic activities related to hippocampal synaptic energy utilization were evaluated, in the perspective of its employment for Dementias and Depression Therapy. Two types of synaptic plasma membranes (SPM1 and SPM2) were isolated from the hippocampus of rats treated with l-acetylcarnitine (30 and 60mg/kg i.p., 28 days, 5 days/week). Acetylcholinesterase (AChE); Na(+), K(+), Mg(2+)-ATP-ase; ouabain-insensitive Mg(2+)-ATP-ase; Na(+), K(+)-ATP-ase; Ca(2+), Mg(2+)-ATP-ase activities were evaluated. In control animals, enzymatic activities were differently expressed in SPM1 , being the evaluated enzymatic activities higher in SPM2. Subchronic treatment with l-acetylcarnitine (i) did not modify AChE on both SPMs; (ii) increased Na(+), K(+), Mg(2+)-ATP-ase, ouabain-insensitive Mg(2+)-ATP-ase and Na(+), K(+)-ATP-ase at the dose of 30 and 60mg/kg on SPM1 and SPM2; (iii) increased Ca(2+), Mg(2+)-ATP-ase activity on both SPMs at the dose of 60mg/kg. These results have been discussed considering the pathophysiology and treatment of Dementias and Depression because, although referred to normal healthy animals, they support the notion that l-acetylcarnitine may have positive effects in these pathologies.

  20. Profiling of the cell surface proteome.

    PubMed

    Jang, Jun Ho; Hanash, Samir

    2003-10-01

    The in depth-mining of the proteome necessitates the comprehensive analysis of proteins in individual subcellular compartments to uncover interesting patterns of protein expression that include assessment of protein location, trafficking and of post-translational modifications that are location specific. One of the compartments of substantial interest from a diagnostic and therapeutic point of view is the plasma membrane which contains intrinsic membrane proteins and other proteins expressed on the cell surface. Technologies are currently available for the comprehensive profiling of the cell surface proteome that rely on protein tagging of intact cells. Studies are emerging that point to unexpected patterns of expression of specific proteins on the cell surface, with a common occurrence of proteins previously considered to occur predominantly in other compartments, notably the endoplasmic reticulum. The profiling of the cell surface and plasma membrane proteomes will likely provide novel insights and uncover disease related alterations. PMID:14625857

  1. A simple solution to expanding available reference materials for Laser Ablation Inductively Coupled Plasma Mass Spectrometry analysis: Applications to sedimentary materials

    NASA Astrophysics Data System (ADS)

    Shaheen, Mohamed E.; Fryer, Brian J.

    2011-08-01

    Analytical data on sediments are of great importance in understanding and documenting environmental issues. For laboratories interested in in-situ chemical analysis of sediments by LA-ICP-MS, a major issue is the lack of appropriate matrix matched sediment reference materials. Those available were largely designed for partial extractions which generally do not reflect the total elemental compositions. In this work we provide a comprehensive study on chemical compositions of seven currently available sediment reference materials (Lake sediments: LKSD-1, LKSD-2, LKSD-3, Stream sediments: STSD-2, STSD-3, and Marine sediments: PACS-2, MESS-3) as determined by Solution Nebulization Inductively Coupled Plasma Mass Spectrometry (SN-ICP-MS) and Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) after digestion in a mixture of concentrated HNO 3 and HF acids. We also report a simple method to prepare these sediment reference materials and more generally appropriate sediment cores for LA-ICP-MS analysis using epoxy resin. This sample preparation method maintains sediment integrity for high spatial resolution analysis which is required for tracing changes in environmental conditions over short time periods. This work also demonstrates the application of fs-LA-ICP-MS as a tool for direct, rapid and high spatial resolution analysis of sediments.

  2. 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.

  3. Metal and metalloid multi-elementary ICP-MS validation in whole blood, plasma, urine and hair. Reference values.

    PubMed

    Goullé, Jean-Pierre; Mahieu, Loïc; Castermant, Julien; Neveu, Nicolas; Bonneau, Laurent; Lainé, Gilbert; Bouige, Daniel; Lacroix, Christian

    2005-10-01

    Four multi-elementary metal and metalloid quantification methods using inductively coupled plasma mass spectrometry (ICP-MS) were developed and validated in human whole blood, plasma, urine and hair by means of a single preparation procedure for each sample. The ICP-MS measurements were performed using a Thermo Elemental X7CCT series and PlasmaLab software without a dynamic reaction cell. With this procedure 27-32 elements can be simultaneously quantified in biological matrices: Li, Be, B, Al, V, Cr, Mn, Co, Ni, Cu, Zn, Ga, Ge, As, Se, Rb, Sr, Mo, Pd, Ag, Cd, Sn, Sb, Te, Ba, W, Pt, Hg, Tl, Pb, Bi, U. Whole blood, plasma and urine samples (0.4 ml each) were diluted with purified water, acid, triton X100 and butanol. Rhodium was used as internal standard. The urine sample results were corrected for enzymatic creatinine determination. Twenty-five milligrams hair samples were acid mineralized after a decontamination procedure and diluted as previously described for biological fluids. To be validated, each element had to show linearity with a correlation coefficient higher than 0.99. The intra-assay and inter-assay inaccuracy, measured as the variation coefficient, were below 5 and 10% respectively. Global performance was assessed by a quality control program. Our laboratory is a registered participant of the Institut National de Santé Publique du Québec (Sainte-Foy, Canada) inter-laboratory comparison program for whole blood, urine, and beard hair of non-occupationally exposed individuals spiked with selected elements. In our study multi-element metal and metalloid analysis was assessed for 27 elements in whole blood, 27 elements in plasma, 30 elements in urine and 32 elements in hair, from 0 to 25, or 250 to 1000 ng/ml, depending on the element. Quantification limits ranged from 0.002 ng/ml (U) to 8.1 ng/ml (Al) for whole blood, from 0.002 ng/ml (U) to 7.7 ng/ml (Al) for plasma, from 0.001 ng/ml (U) to 2.2 ng/ml (Se) for urine, and from 0.2 pg/mg (Tl) to 0.5 ng

  4. 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.

  5. Reference measurements for total mercury and methyl mercury content in marine biota samples using direct or species-specific isotope dilution inductively coupled plasma mass spectrometry.

    PubMed

    Krata, Agnieszka; Vassileva, Emilia; Bulska, Ewa

    2016-11-01

    The analytical procedures for reference measurements of the total Hg and methyl mercury (MeHg) mass fractions at various concentration levels in marine biota samples, candidates for certified reference materials (oyster and clam Gafrarium tumidum), were evaluated. Two modes of application of isotope dilution inductively coupled plasma mass spectrometry method (ID ICP-MS), namely direct isotope dilution and species-specific isotope dilution analysis with the use of two different quantification mass spectrometry techniques were compared. The entire ID ICP-MS measurement procedure was described by mathematical modelling and the combined uncertainty of measurement results was estimated. All factors influencing the final results as well as isotopic equilibrium were systematically investigated. This included the procedural blank, the moisture content in the biota samples and all factors affecting the blend ratio measurements (instrumental background, spectral interferences, dead time and mass discrimination effects as well as the repeatability of measured isotopic ratios). Modelling of the entire measurement procedures and the use of appropriate certified reference materials enable to assure the traceability of obtained values to the International System of Units (SI): the mole or the kilogram. The total mass fraction of mercury in oyster and clam biota samples, after correction for moisture contents, was found to be: 21.1 (1.1) 10(-9) kg kg(-1) (U =5.1% relative, k=2) and 390.0 (9.4) 10(-9) kg kg(-1) (U=2.4% relative, k=2), respectively. For the determination of mercury being present as methyl mercury, the non-chromatographic separation on anion-exchange resin AG1-X8 of the blended samples was applied. The content of MeHg (as Hg) in oyster sample was found: 4.81 (24) 10(-9)kgkg(-1) (U=5.0%, k=2) and 4.84 (21) 10(-9)kgkg(-1) (U=4.3%, k=2) with the use of quadrupole (ICP QMS) or sector field (ICP SFMS) inductively coupled plasma mass spectrometers, respectively. In the

  6. Reference measurements for total mercury and methyl mercury content in marine biota samples using direct or species-specific isotope dilution inductively coupled plasma mass spectrometry.

    PubMed

    Krata, Agnieszka; Vassileva, Emilia; Bulska, Ewa

    2016-11-01

    The analytical procedures for reference measurements of the total Hg and methyl mercury (MeHg) mass fractions at various concentration levels in marine biota samples, candidates for certified reference materials (oyster and clam Gafrarium tumidum), were evaluated. Two modes of application of isotope dilution inductively coupled plasma mass spectrometry method (ID ICP-MS), namely direct isotope dilution and species-specific isotope dilution analysis with the use of two different quantification mass spectrometry techniques were compared. The entire ID ICP-MS measurement procedure was described by mathematical modelling and the combined uncertainty of measurement results was estimated. All factors influencing the final results as well as isotopic equilibrium were systematically investigated. This included the procedural blank, the moisture content in the biota samples and all factors affecting the blend ratio measurements (instrumental background, spectral interferences, dead time and mass discrimination effects as well as the repeatability of measured isotopic ratios). Modelling of the entire measurement procedures and the use of appropriate certified reference materials enable to assure the traceability of obtained values to the International System of Units (SI): the mole or the kilogram. The total mass fraction of mercury in oyster and clam biota samples, after correction for moisture contents, was found to be: 21.1 (1.1) 10(-9) kg kg(-1) (U =5.1% relative, k=2) and 390.0 (9.4) 10(-9) kg kg(-1) (U=2.4% relative, k=2), respectively. For the determination of mercury being present as methyl mercury, the non-chromatographic separation on anion-exchange resin AG1-X8 of the blended samples was applied. The content of MeHg (as Hg) in oyster sample was found: 4.81 (24) 10(-9)kgkg(-1) (U=5.0%, k=2) and 4.84 (21) 10(-9)kgkg(-1) (U=4.3%, k=2) with the use of quadrupole (ICP QMS) or sector field (ICP SFMS) inductively coupled plasma mass spectrometers, respectively. In the

  7. The human urinary proteome contains more than 1500 proteins, including a large proportion of membrane proteins

    PubMed Central

    Adachi, Jun; Kumar, Chanchal; Zhang, Yanling; Olsen, Jesper V; Mann, Matthias

    2006-01-01

    Background Urine is a desirable material for the diagnosis and classification of diseases because of the convenience of its collection in large amounts; however, all of the urinary proteome catalogs currently being generated have limitations in their depth and confidence of identification. Our laboratory has developed methods for the in-depth characterization of body fluids; these involve a linear ion trap-Fourier transform (LTQ-FT) and a linear ion trap-orbitrap (LTQ-Orbitrap) mass spectrometer. Here we applied these methods to the analysis of the human urinary proteome. Results We employed one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and reverse phase high-performance liquid chromatography for protein separation and fractionation. Fractionated proteins were digested in-gel or in-solution, and digests were analyzed with the LTQ-FT and LTQ-Orbitrap at parts per million accuracy and with two consecutive stages of mass spectrometric fragmentation. We identified 1543 proteins in urine obtained from ten healthy donors, while essentially eliminating false-positive identifications. Surprisingly, nearly half of the annotated proteins were membrane proteins according to Gene Ontology (GO) analysis. Furthermore, extracellular, lysosomal, and plasma membrane proteins were enriched in the urine compared with all GO entries. Plasma membrane proteins are probably present in urine by secretion in exosomes. Conclusion Our analysis provides a high-confidence set of proteins present in human urinary proteome and provides a useful reference for comparing datasets obtained using different methodologies. The urinary proteome is unexpectedly complex and may prove useful in biomarker discovery in the future. PMID:16948836

  8. 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. PMID:25000688

  9. Quantitative transporter proteomics by liquid chromatography with tandem mass spectrometry: addressing methodologic issues of plasma membrane isolation and expression-activity relationship.

    PubMed

    Kumar, Vineet; Prasad, Bhagwat; Patilea, Gabriela; Gupta, Anshul; Salphati, Laurent; Evers, Raymond; Hop, Cornelis E C A; Unadkat, Jashvant D

    2015-02-01

    To predict transporter-mediated drug disposition using physiologically based pharmacokinetic models, one approach is to measure transport activity and relate it to protein expression levels in cell lines (overexpressing the transporter) and then scale these to via in vitro to in vivo extrapolation (IVIVE). This approach makes two major assumptions. First, that the expression of the transporter is predominantly in the plasma membrane. Second, that there is a linear correlation between expression level and activity of the transporter protein. The present study was conducted to test these two assumptions. We evaluated two commercially available kits that claimed to separate plasma membrane from other cell membranes. The Qiagen Qproteome kit yielded very little protein in the fraction purported to be the plasma membrane. The Abcam Phase Separation kit enriched the plasma membrane but did not separate it from other intracellular membranes. For the Abcam method, the expression level of organic anion-transporting polypeptides (OATP) 1B1/2B1 and breast cancer resistance protein (BCRP) proteins in all subcellular fractions isolated from cells or human liver tissue tracked that of Na⁺-K⁺ ATPase. Assuming that Na⁺-K⁺ ATPase is predominantly located in the plasma membrane, these data suggest that the transporters measured are also primarily located in the plasma membrane. Using short hairpin RNA, we created clones of cell lines with varying degrees of OATP1B1 or BCRP expression level. In these clones, transport activity of OATP1B1 or BCRP was highly correlated with protein expression level (r² > 0.9). These data support the use of transporter expression level data and activity data from transporter overexpressing cell lines for IVIVE of transporter-mediated disposition of drugs.

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

    PubMed

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

    2015-06-01

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

  11. 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.

  12. [Variability of healthy human proteome].

    PubMed

    Pakharukova, N A; Pastushkova, L Kh; Moshkovskiĭ, S A; Larina, I M

    2012-01-01

    The purpose of this review is to analyze investigations devoted to characteristic of protein variability and diversity of their posttranslational modifications in healthy humans. The numerous researches have demonstrated that proteomic profile has a considerable both intra- and inter-individual variability, and quite often normal variability of some proteins can be comparable to changes observed in pathological processes. Results obtained by our research group have confirmed high intra-individual variability of serum low-molecular subproteome of healthy volunteers, certified by a special medial committee. Proteins characterized by high variability in normal conditions (e.g. haptoglobin--0-40 mg/ml; lysozyme--0,01-0,1 mg/ml; C-reactive protein--0,01-0,3 mg/ml) should be excluded from the list of potential biomarkers. On the contrary, proteins and peptides characterized by insignificant dispersion in healthy population (such as albumin--coefficient of variation (CV) 9%; transferrin--CV14%; C3c complement--CV 17%, alpha-1 acid glycoprotein--CV 21%, alpha2-macroglobulin--CV 20%; transthyretin fragment--CV 28,3% and beta-chain alpha2-HS-glycoprotein--CV 29,7%) can provide us with important information about state of health. Thus investigations of plasticity in proteomic profiles of healthy humans will help to correct reference intervals used in clinical proteomics. PMID:23289293

  13. Measurement of vanadium, nickel, and arsenic in seawater and urine reference materials by inductively coupled plasma mass spectrometry with cryogenic desolvation

    SciTech Connect

    Alves, L.C.; Allen, L.A.; Houk, R.S. Iowa State Univ., Ames, IA )

    1993-09-15

    Addition of a small dose (2%) of H[sub 2] to the aerosol gas flow enhanced analyte signals by a factor of 2-3, which compensated for the loss of analyte signal that accompanied earlier efforts at cryogenic desolvation with inductively coupled plasma mass spectrometry (ICP-MS). Vanadium, nickel, and arsenic at microgram per liter levels in urine, river, and seawater reference materials were determined. The polyatomic ions ClO[sup +], CaO[sup +], and ArCl[sup +], which normally cause severe overlap interferences for these elements, were attenuated to manageable levels by cryogenic desolvation. The samples were simply diluted with 1% HNO[sub 2] so that the chloride could be removed as HCl. The analytical results obtained for these standard reference materials agreed closely with the certified or recommended values. The detection limit ranges (3[sigma]) obtained were 10-1000 ng L[sup [minus]1] for V, 0.03-20 [mu]g L[sup [minus]1] for Ni, and 4-7000 ng L[sup [minus]1] for As in the original samples. The samples were introduced by flow injection to minimize clogging of the sampling orifice. 29 refs., 2 figs., 5 tabs.

  14. 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.

  15. Differential Plasma Glycoproteome of p19ARF Skin Cancer Mouse Model Using the Corra Label-Free LC-MS Proteomics Platform

    PubMed Central

    Letarte, Simon; Brusniak, Mi-Youn; Campbell, David; Eddes, James; Kemp, Christopher J.; Lau, Hollis; Mueller, Lukas; Schmidt, Alexander; Shannon, Paul; Kelly-Spratt, Karen S.; Vitek, Olga; Zhang, Hui; Aebersold, Ruedi; Watts, Julian D.

    2010-01-01

    A proof-of-concept demonstration of the use of label-free quantitative glycoproteomics for biomarker discovery workflow is presented here, using a mouse model for skin cancer as an example. Blood plasma was collected from 10 control mice, and 10 mice having a mutation in the p19ARF gene, conferring them high propensity to develop skin cancer after carcinogen exposure. We enriched for N-glycosylated plasma proteins, ultimately generating deglycosylated forms of the modified tryptic peptides for liquid chromatography mass spectrometry (LC-MS) analyses. LC-MS runs for each sample were then performed with a view to identifying proteins that were differentially abundant between the two mouse populations. We then used a recently developed computational framework, Corra, to perform peak picking and alignment, and to compute the statistical significance of any observed changes in individual peptide abundances. Once determined, the most discriminating peptide features were then fragmented and identified by tandem mass spectrometry with the use of inclusion lists. We next assessed the identified proteins to see if there were sets of proteins indicative of specific biological processes that correlate with the presence of disease, and specifically cancer, according to their functional annotations. As expected for such sick animals, many of the proteins identified were related to host immune response. However, a significant number of proteins also directly associated with processes linked to cancer development, including proteins related to the cell cycle, localisation, trasport, and cell death. Additional analysis of the same samples in profiling mode, and in triplicate, confirmed that replicate MS analysis of the same plasma sample generated less variation than that observed between plasma samples from different individuals, demonstrating that the reproducibility of the LC-MS platform was sufficient for this application. These results thus show that an LC-MS-based workflow

  16. Systems proteomics of liver mitochondria function.

    PubMed

    Williams, Evan G; Wu, Yibo; Jha, Pooja; Dubuis, Sébastien; Blattmann, Peter; Argmann, Carmen A; Houten, Sander M; Amariuta, Tiffany; Wolski, Witold; Zamboni, Nicola; Aebersold, Ruedi; Auwerx, Johan

    2016-06-10

    Recent improvements in quantitative proteomics approaches, including Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS), permit reproducible large-scale protein measurements across diverse cohorts. Together with genomics, transcriptomics, and other technologies, transomic data sets can be generated that permit detailed analyses across broad molecular interaction networks. Here, we examine mitochondrial links to liver metabolism through the genome, transcriptome, proteome, and metabolome of 386 individuals in the BXD mouse reference population. Several links were validated between genetic variants toward transcripts, proteins, metabolites, and phenotypes. Among these, sequence variants in Cox7a2l alter its protein's activity, which in turn leads to downstream differences in mitochondrial supercomplex formation. This data set demonstrates that the proteome can now be quantified comprehensively, serving as a key complement to transcriptomics, genomics, and metabolomics--a combination moving us forward in complex trait analysis. PMID:27284200

  17. 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. PMID:21394846

  18. Systems proteomics of liver mitochondria function.

    PubMed

    Williams, Evan G; Wu, Yibo; Jha, Pooja; Dubuis, Sébastien; Blattmann, Peter; Argmann, Carmen A; Houten, Sander M; Amariuta, Tiffany; Wolski, Witold; Zamboni, Nicola; Aebersold, Ruedi; Auwerx, Johan

    2016-06-10

    Recent improvements in quantitative proteomics approaches, including Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS), permit reproducible large-scale protein measurements across diverse cohorts. Together with genomics, transcriptomics, and other technologies, transomic data sets can be generated that permit detailed analyses across broad molecular interaction networks. Here, we examine mitochondrial links to liver metabolism through the genome, transcriptome, proteome, and metabolome of 386 individuals in the BXD mouse reference population. Several links were validated between genetic variants toward transcripts, proteins, metabolites, and phenotypes. Among these, sequence variants in Cox7a2l alter its protein's activity, which in turn leads to downstream differences in mitochondrial supercomplex formation. This data set demonstrates that the proteome can now be quantified comprehensively, serving as a key complement to transcriptomics, genomics, and metabolomics--a combination moving us forward in complex trait analysis.

  19. 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.

  20. 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

    2014-03-01

    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. PMID:24500811

  1. 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

  2. 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

  3. 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

  4. Human fallopian tube proteome shows high coverage of mesenchymal stem cells associated proteins.

    PubMed

    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

  5. 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.

  6. 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.

  7. Approaching clinical proteomics: current state and future fields of application in fluid proteomics.

    PubMed

    Apweiler, Rolf; Aslanidis, Charalampos; Deufel, Thomas; Gerstner, Andreas; Hansen, Jens; Hochstrasser, Dennis; Kellner, Roland; Kubicek, Markus; Lottspeich, Friedrich; Maser, Edmund; Mewes, Hans-Werner; Meyer, Helmut E; Müllner, Stefan; Mutter, Wolfgang; Neumaier, Michael; Nollau, Peter; Nothwang, Hans G; Ponten, Fredrik; Radbruch, Andreas; Reinert, Knut; Rothe, Gregor; Stockinger, Hannes; Tarnok, Attila; Taussig, Mike J; Thiel, Andreas; Thiery, Joachim; Ueffing, Marius; Valet, Günther; Vandekerckhove, Joel; Verhuven, Wiltrud; Wagener, Christoph; Wagner, Oswald; Schmitz, Gerd

    2009-01-01

    The field of clinical proteomics offers opportunities to identify new disease biomarkers in body fluids, cells and tissues. These biomarkers can be used in clinical applications for diagnosis, stratification of patients for specific treatment, or therapy monitoring. New protein array formats and improved spectrometry technologies have brought these analyses to a level with potential for use in clinical diagnostics. The nature of the human body fluid proteome with its large dynamic range of protein concentrations presents problems with quantitation. The extreme complexity of the proteome in body fluids presents enormous challenges and requires the establishment of standard operating procedures for handling of specimens, increasing sensitivity for detection and bioinformatical tools for distribution of proteomic data into the public domain. From studies of in vitro diagnostics, especially in clinical chemistry, it is evident that most errors occur in the preanalytical phase and during implementation of the diagnostic strategy. This is also true for clinical proteomics, and especially for fluid proteomics because of the multiple pretreatment processes. These processes include depletion of high-abundance proteins from plasma or enrichment processes for urine where biological variation or differences in proteolytic activities in the sample along with preanalytical variables such as inter- and intra-assay variability will likely influence the results of proteomics studies. However, before proteomic analysis can be introduced at a broader level into the clinical setting, standardization of the preanalytical phase including patient preparation, sample collection, sample preparation, sample storage, measurement and data analysis needs to be improved. In this review, we discuss the recent technological advances and applications that fulfil the criteria for clinical proteomics, with the focus on fluid proteomics. These advances relate to preanalytical factors, analytical

  8. 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

  9. Proteomics data repositories

    PubMed Central

    Riffle, Michael; Eng, Jimmy K.

    2010-01-01

    The field of proteomics, particularly the application of mass spectrometry analysis to protein samples, is well-established and growing rapidly. Proteomics studies generate large volumes of raw experimental data and inferred biological results. To facilitate the dissemination of these data, centralized data repositories have been developed that make the data and results accessible to proteomics researchers and biologists alike. This review of proteomics data repositories focuses exclusively on freely-available, centralized data resources that disseminate or store experimental mass spectrometry data and results. The resources chosen reflect a current “snapshot” of the state of resources available with an emphasis placed on resources that may be of particular interest to yeast researchers. Resources are described in terms of their intended purpose and the features and functionality provided to users. PMID:19795424

  10. 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. PMID:25919366

  11. 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

  12. 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.

  13. Proteomics Research in Schizophrenia

    PubMed Central

    Davalieva, Katarina; Maleva Kostovska, Ivana; Dwork, Andrew J.

    2016-01-01

    Despite intense scientific efforts, the neuropathology and pathophysiology of schizophrenia are poorly understood. Proteomic studies, by testing large numbers of proteins for associations with disease, may contribute to the understanding of the molecular mechanisms of schizophrenia. They may also indicate the types and locations of cells most likely to harbor pathological alterations. Investigations using proteomic approaches have already provided much information on quantitative and qualitative protein patterns in postmortem brain tissue, peripheral tissues and body fluids. Different proteomic technologies such as 2-D PAGE, 2-D DIGE, SELDI-TOF, shotgun proteomics with label-based (ICAT), and label-free (MSE) quantification have been applied to the study of schizophrenia for the past 15 years. This review summarizes the results, mostly from brain but also from other tissues and bodily fluids, of proteomics studies in schizophrenia. Emphasis is given to proteomics platforms, varying sources of material, proposed candidate biomarkers emerging from comparative proteomics studies, and the specificity of the putative markers in terms of other mental illnesses. We also compare proteins altered in schizophrenia with reports of protein or mRNA sequences that are relatively enriched in specific cell types. While proteomic studies of schizophrenia find abnormalities in the expression of many proteins that are not cell type-specific, there appears to be a disproportionate representation of proteins whose synthesis and localization are highly enriched in one or more brain cell type compared with other types of brain cells. Two of the three proteins most commonly altered in schizophrenia are aldolase C and glial fibrillary acidic protein, astrocytic proteins with entirely different functions, but the studies are approximately evenly divided with regard to the direction of the differences and the concordance or discordance between the two proteins. Alterations of common myelin

  14. Advanced proteomic liquid chromatography

    PubMed Central

    Xie, Fang; Smith, Richard D.; Shen, Yufeng

    2012-01-01

    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. PMID:22840822

  15. Nanoscaled Proteomic Analysis

    NASA Astrophysics Data System (ADS)

    Xu, Yan; Jia, Lee

    2013-09-01

    Global proteomics research is currently hampered by the extremely complexity of the proteome and the absence of techniques like the polymerase chain reaction in genomics which enables multiplication of a single protein molecule. Since all the existing analytical technologies cannot overcome the detection limit and the dynamic concentration barrier, development of improved analytical technologies at nanoscale, ideally those that could recognize single protein molecule in the presence of high abundant of others, is a high priority for proteomics. In this chapter, we will show the state-of-the-art of nanoproteomics, i.e., the application of nanotechnologies to proteomics. Various nanomaterials including carbon nanomaterials, magnetic nanoparticles, silica nanoparticles, polymer and copolymer nanoparticles, metal and metal oxide nanoparticles have been used to improve sensitivity, specificity, and repeatability of proteomic analysis especially when the multidimensional separation system coupled with MALDI-TOF-MS is used. Among them, gold nanoparticles (GNPs) and carbon nanotubes (CNTs) are the two most important nanomaterials: while GNPs are frequently utilized for enzyme immobilization, high throughput bioassay, selection of target-peptides and target-protein, CNTs including single-walled carbon nanotubes (SWCNTs) and mutiple-walled carbon nanotubes (MWCNTs) have wide applications to electronic sensor, sensitive immunodetection, nanobiocatalysis, affinity probes, MALDI matrices, protein digestion, peptides enrichment and analysis. In perspectives, a deep understanding of the structures and property of nanomaterials and interdisciplinary applications of nanotechnology to proteomics will certainly be revolutionary and intellectually rewarding.

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

    PubMed

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

    2016-09-01

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

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

    PubMed

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

    2016-09-01

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

  18. Collaborations in Proteomics Research - Office of Cancer Clinical Proteomics Research

    Cancer.gov

    The National Cancer Institute (NCI), through the Office of Cancer Clinical Proteomics Research (OCCPR), has signed two Memorandums of Understanding (MOUs) in the sharing of proteomics reagents and protocols

  19. Introduction to mass spectrometry-based proteomics.

    PubMed

    Matthiesen, Rune; Bunkenborg, Jakob

    2013-01-01

    Mass spectrometry has been widely applied to study biomolecules and one rapidly developing field is the global analysis of proteins, proteomics. Understanding and handling mass spectrometry data is a multifaceted task that requires many decisions to be made to get the most comprehensive information from an experiment. Later chapters in this book deal in-depth with various aspects of the process and how different tools can be applied to the many analytical challenges. This introductory chapter is intended as a basic introduction to mass spectrometry (MS)-based proteomics to set the scene for newcomers and give pointers to reference material. There are many applications of mass spectrometry in proteomics and each application is associated with some analytical choices, instrumental limitations and data processing steps that depend on the aim of the study and means of conducting it. Different aspects of the proteome can be explored by choosing the right combination of sample preparation, MS instrumentation and data processing. This chapter gives an outline for some of these commonly used setups and some of the key concepts, many of which are explored in greater depth in later chapters. PMID:23666720

  20. Role of Proteomics in Crop Stress Tolerance.

    PubMed

    Ahmad, Parvaiz; Abdel Latef, Arafat A H; Rasool, Saiema; Akram, Nudrat A; Ashraf, Muhammad; Gucel, Salih

    2016-01-01

    Plants often experience various biotic and abiotic stresses during their life cycle. The abiotic stresses include mainly drought, salt, temperature (low/high), flooding and nutritional deficiency/excess which hamper crop growth and yield to a great extent. In view of a projection 50% of the crop loss is attributable to abiotic stresses. However, abiotic stresses cause a myriad of changes in physiological, molecular and biochemical processes operating in plants. It is now widely reported that several proteins respond to these stresses at pre- and post-transcriptional and translational levels. By knowing the role of these stress inducible proteins, it would be easy to comprehensively expound the processes of stress tolerance in plants. The proteomics study offers a new approach to discover proteins and pathways associated with crop physiological and stress responses. Thus, studying the plants at proteomic levels could help understand the pathways involved in stress tolerance. Furthermore, improving the understanding of the identified key metabolic proteins involved in tolerance can be implemented into biotechnological applications, regarding recombinant/transgenic formation. Additionally, the investigation of identified metabolic processes ultimately supports the development of antistress strategies. In this review, we discussed the role of proteomics in crop stress tolerance. We also discussed different abiotic stresses and their effects on plants, particularly with reference to stress-induced expression of proteins, and how proteomics could act as vital biotechnological tools for improving stress tolerance in plants. PMID:27660631

  1. Role of Proteomics in Crop Stress Tolerance

    PubMed Central

    Ahmad, Parvaiz; Abdel Latef, Arafat A. H.; Rasool, Saiema; Akram, Nudrat A.; Ashraf, Muhammad; Gucel, Salih

    2016-01-01

    Plants often experience various biotic and abiotic stresses during their life cycle. The abiotic stresses include mainly drought, salt, temperature (low/high), flooding and nutritional deficiency/excess which hamper crop growth and yield to a great extent. In view of a projection 50% of the crop loss is attributable to abiotic stresses. However, abiotic stresses cause a myriad of changes in physiological, molecular and biochemical processes operating in plants. It is now widely reported that several proteins respond to these stresses at pre- and post-transcriptional and translational levels. By knowing the role of these stress inducible proteins, it would be easy to comprehensively expound the processes of stress tolerance in plants. The proteomics study offers a new approach to discover proteins and pathways associated with crop physiological and stress responses. Thus, studying the plants at proteomic levels could help understand the pathways involved in stress tolerance. Furthermore, improving the understanding of the identified key metabolic proteins involved in tolerance can be implemented into biotechnological applications, regarding recombinant/transgenic formation. Additionally, the investigation of identified metabolic processes ultimately supports the development of antistress strategies. In this review, we discussed the role of proteomics in crop stress tolerance. We also discussed different abiotic stresses and their effects on plants, particularly with reference to stress-induced expression of proteins, and how proteomics could act as vital biotechnological tools for improving stress tolerance in plants.

  2. Role of Proteomics in Crop Stress Tolerance

    PubMed Central

    Ahmad, Parvaiz; Abdel Latef, Arafat A. H.; Rasool, Saiema; Akram, Nudrat A.; Ashraf, Muhammad; Gucel, Salih

    2016-01-01

    Plants often experience various biotic and abiotic stresses during their life cycle. The abiotic stresses include mainly drought, salt, temperature (low/high), flooding and nutritional deficiency/excess which hamper crop growth and yield to a great extent. In view of a projection 50% of the crop loss is attributable to abiotic stresses. However, abiotic stresses cause a myriad of changes in physiological, molecular and biochemical processes operating in plants. It is now widely reported that several proteins respond to these stresses at pre- and post-transcriptional and translational levels. By knowing the role of these stress inducible proteins, it would be easy to comprehensively expound the processes of stress tolerance in plants. The proteomics study offers a new approach to discover proteins and pathways associated with crop physiological and stress responses. Thus, studying the plants at proteomic levels could help understand the pathways involved in stress tolerance. Furthermore, improving the understanding of the identified key metabolic proteins involved in tolerance can be implemented into biotechnological applications, regarding recombinant/transgenic formation. Additionally, the investigation of identified metabolic processes ultimately supports the development of antistress strategies. In this review, we discussed the role of proteomics in crop stress tolerance. We also discussed different abiotic stresses and their effects on plants, particularly with reference to stress-induced expression of proteins, and how proteomics could act as vital biotechnological tools for improving stress tolerance in plants. PMID:27660631

  3. A proteomic approach to porcine saliva.

    PubMed

    Gutiérrez, Ana M; Cerón, José J; Fuentes-Rubio, María; Tecles, Fernando; Beeley, Josie A

    2014-02-01

    This paper reviews recent progress in salivary animal proteomics, with special reference to the porcine proteome. Until fairly recently, most studies on saliva as a diagnostic fluid have focused on humans, primates and rodents, and the development of salivary analysis in monitoring health in farm animals including pigs has received only limited consideration. The porcine salivary proteome has been characterised by 2D-electrophoresis followed by mass spectrometry. Major and minor proteins have been identified. The use of saliva as a non-invasive biological fluid in monitoring health and disease in pigs will be reviewed, together with the potential use of proteomics for the development of biomarkers. In this review, methods of collection and the composition of porcine saliva will be considered, together with saliva handling and analysis. The overall findings indicate that there is considerable potential for the development of salivary analysis as a non-invasive diagnostic fluid in the pig, and that it offers advantages over other body fluids in this animal.

  4. Introduction to mass spectrometry-based proteomics.

    PubMed

    Matthiesen, Rune; Bunkenborg, Jakob

    2013-01-01

    Mass spectrometry has been widely applied to study biomolecules and one rapidly developing field is the global analysis of proteins, proteomics. Understanding and handling mass spectrometry data is a multifaceted task that requires many decisions to be made to get the most comprehensive information from an experiment. Later chapters in this book deal in-depth with various aspects of the process and how different tools can be applied to the many analytical challenges. This introductory chapter is intended as a basic introduction to mass spectrometry (MS)-based proteomics to set the scene for newcomers and give pointers to reference material. There are many applications of mass spectrometry in proteomics and each application is associated with some analytical choices, instrumental limitations and data processing steps that depend on the aim of the study and means of conducting it. Different aspects of the proteome can be explored by choosing the right combination of sample preparation, MS instrumentation and data processing. This chapter gives an outline for some of these commonly used setups and some of the key concepts, many of which are explored in greater depth in later chapters.

  5. Proteomics analysis of human oligodendroglioma proteome.

    PubMed

    Khaghani-Razi-Abad, Solmaz; Hashemi, Mehrdad; Pooladi, Mehdi; Entezari, Maliheh; Kazemi, Elham

    2015-09-10

    Proteomics analyses enable the identification and quantitation of proteins. From a purely clinical perspective, the application of proteomics based on innovations, may greatly affect the future management of malignant brain tumors. This optimism is based on four main reasons: diagnosis, prognosis, selection of targeted therapy based on molecular profile of the brain tumor and monitoring therapeutic response, or resistance. We extracted the proteins of tumor and normal brain tissues, and then evaluated the protein purity by Bradford test. In this study, we separated the proteins by two-dimensional (2DG) gel electrophoresis methods. Then spots were analyzed, compared using statistical data and specific software and were identified by pH isoelectric, molecular weights and data banks. The protein profiles were determined using 2D gel electrophoresis and MALDI TOF/TOF mass spectrometry approaches. Simple statistical tests were used to establish a putative hierarchy in which the change in protein level was ranked according to a cut-off point with p<0.05. The 2D gel showed a total of 1328 spots among which 157 spots were under-expressed and 276 spots were overexpressed. Most proteins are subjects to post-translational modifications, where amino acid residues may be chemically modified or conjugated by small proteins like ubiquitin. Proteomics is a powerful way to identifying multiple proteins which are altered following a neuropharmacological intervention in a CNS disease. PMID:26002447

  6. Leveraging Genomics Software to Improve Proteomics Results

    SciTech Connect

    Fodor, I K; Nelson, D O

    2005-09-06

    Rigorous data analysis techniques are essential in quantifying the differential expression of proteins in biological samples of interest. Statistical methods from the microarray literature were applied to the analysis of two-dimensional difference gel electrophoresis (2-D DIGE) proteomics experiments, in the context of technical variability studies involving human plasma. Protein expression measurements were corrected to account for observed intensity-dependent biases within gels, and normalized to mitigate observed gel to gel variations. The methods improved upon the results achieved using the best currently available 2-D DIGE proteomics software. The spot-wise protein variance was reduced by 10% and the number of apparently differentially expressed proteins was reduced by over 50%.

  7. Proteomic Assessment of Poultry Spermatozoa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fully characterizing the protein composition of spermatozoa is the first step in utilizing proteomics to delineate the function of sperm proteins. To date, sperm proteome maps have been partially developed for the human, mouse, rat, bull and several invertebrates. Here we report the first proteomic...

  8. Beer and wort proteomics.

    PubMed

    Iimure, Takashi; Kihara, Makoto; Sato, Kazuhiro

    2014-01-01

    Proteome analysis provides a way to identify proteins related to the quality traits of beer. A number of protein species in beer and wort have been identified by two-dimensional gel electrophoresis combined with enzyme digestion such as trypsin, followed by mass spectrometry analyses and/or liquid chromatography mass/mass spectrometry. In addition, low molecular weight polypeptides in beer have been identified by the combination of non-enzyme digestion and mass analyses. These data sets of various molecular weight polypeptides (i.e., proteomes) provide a platform for analyzing protein functions in beer. Several novel proteins related to beer quality traits such as foam stability and haze formation have been identified by analyzing these proteomes. Some of the proteins have been applied to the development of efficient protein or DNA markers for trait selection in malting barley breeding. In this chapter, recent proteome studies of beer and wort are reviewed, and the methods and protocols of beer and wort proteome analysis are described.

  9. The Cysteine Proteome

    PubMed Central

    Go, Young-Mi; Chandler, Joshua D.; Jones, Dean P.

    2015-01-01

    The cysteine (Cys) proteome is a major component of the adaptive interface between the genome and the exposome. The thiol moiety of Cys undergoes a range of biologic modifications enabling biological switching of structure and reactivity. These biological modifications include sulfenylation and disulfide formation, formation of higher oxidation states, S-nitrosylation, persulfidation, metallation, and other modifications. Extensive knowledge about these systems and their compartmentalization now provides a foundation to develop advanced integrative models of Cys proteome regulation. In particular, detailed understanding of redox signaling pathways and sensing networks is becoming available to discriminate network structures. This research focuses attention on the need for atlases of Cys modifications to develop systems biology models. Such atlases will be especially useful for integrative studies linking the Cys proteome to imaging and other omics platforms, providing a basis for improved redox-based therapeutics. Thus, a framework is emerging to place the Cys proteome as a complement to the quantitative proteome in the omics continuum connecting the genome to the exposome. PMID:25843657

  10. Environmental proteomics and metallomics.

    PubMed

    López-Barea, Juan; Gómez-Ariza, José Luis

    2006-04-01

    Monitoring environmental pollution using biomarkers requires detailed knowledge about the markers, and many only allow a partial assessment of pollution. New proteomic methods (environmental proteomics) can identify proteins that, after validation, might be useful as alternative biomarkers, although this approach also has its limitations, derived mainly from their application to non-model organisms. Initial studies using environmental proteomics were carried out in animals exposed to model pollutants, and led to the concept of protein expression signatures. Experiments have been carried out in model organisms (yeast, Arabidopsis, rat cells, or mice) exposed to model contaminants. Over the last few years, proteomics has been applied to organisms from ecosystems with different pollution levels, forming the basis of an environmental branch in proteomics. Another focus is connected with the presence of metals bound to biomolecules, which adds an additional dimension to metal-biomolecule and metalloprotein characterization - the field of metallomics. The metallomic approach considers the metallome: a whole individual metal or metalloid species within a cell or tissue. A metallomic analytical approach (MAA) is proposed as a new tool to study and identify metalloproteins.

  11. Proteomics in alcohol research.

    PubMed

    Anni, Helen; Israel, Yedy

    2002-01-01

    The proteome is the complete set of proteins in an organism. It is considerably larger and more complex than the genome--the collection of genes that encodes these proteins. Proteomics deals with the qualitative and quantitative study of the proteome under physiological and pathological conditions (e.g., after exposure to alcohol, which causes major changes in numerous proteins of different cell types). To map large proteomes such as the human proteome, proteins from discrete tissues, cells, cell components, or biological fluids are first separated by high-resolution two-dimensional electrophoresis and multidimensional liquid chromatography. Then, individual proteins are identified by mass spectrometry. The huge amount of data acquired using these techniques is analyzed and assembled by fast computers and bioinformatics tools. Using these methods, as well as other technological advances, alcohol researchers can gain a better understanding of how alcohol globally influences protein structure and function, protein-protein interactions, and protein networks. This knowledge ultimately will assist in the early diagnosis and prognosis of alcoholism and the discovery of new drug targets and medications for treatment.

  12. Accuracy and Reproducibility in Quantification of Plasma Protein Concentrations by Mass Spectrometry without the Use of Isotopic Standards

    PubMed Central

    Kramer, Gertjan; Woolerton, Yvonne; van Straalen, Jan P.; Vissers, Johannes P. C.; Dekker, Nick; Langridge, James I.; Beynon, Robert J.; Speijer, Dave; Sturk, Auguste; Aerts, Johannes M. F. G.

    2015-01-01

    Background Quantitative proteomic analysis with mass spectrometry holds great promise for simultaneously quantifying proteins in various biosamples, such as human plasma. Thus far, studies addressing the reproducible measurement of endogenous protein concentrations in human plasma have focussed on targeted analyses employing isotopically labelled standards. Non-targeted proteomics, on the other hand, has been less employed to this end, even though it has been instrumental in discovery proteomics, generating large datasets in multiple fields of research. Results Using a non-targeted mass spectrometric assay (LCMSE), we quantified abundant plasma proteins (43 mg/mL—40 ug/mL range) in human blood plasma specimens from 30 healthy volunteers and one blood serum sample (ProteomeXchange: PXD000347). Quantitative results were obtained by label-free mass spectrometry using a single internal standard to estimate protein concentrations. This approach resulted in quantitative results for 59 proteins (cut off ≥11 samples quantified) of which 41 proteins were quantified in all 31 samples and 23 of these with an inter-assay variability of ≤ 20%. Results for 7 apolipoproteins were compared with those obtained using isotope-labelled standards, while 12 proteins were compared to routine immunoassays. Comparison of quantitative data obtained by LCMSE and immunoassays showed good to excellent correlations in relative protein abundance (r = 0.72–0.96) and comparable median concentrations for 8 out of 12 proteins tested. Plasma concentrations of 56 proteins determined by LCMSE were of similar accuracy as those reported by targeted studies and 7 apolipoproteins quantified by isotope-labelled standards, when compared to reference concentrations from literature. Conclusions This study shows that LCMSE offers good quantification of relative abundance as well as reasonable estimations of concentrations of abundant plasma proteins. PMID:26474480

  13. Establishing Substantial Equivalence: Proteomics

    NASA Astrophysics Data System (ADS)

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

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

  14. The proteomics quantification dilemma.

    PubMed

    Jungblut, Peter R

    2014-07-31

    Proteomics is dominated today by the protein expression discourse, which favorites the bottom-up approach because of its high throughput and its high sensitivity. For quantification this proceeding is misleading, if a protein is present with more than one protein species in the sample to be analyzed. The protein speciation discourse considers this more realistic situation and affords the top-down procedures or at least a separation of the protein species in advance to identification and quantification. Today all of the top-down procedures are one order of magnitude less sensitive than the bottom-up ones. To increase sensitivity and to increase throughput are major challenges for proteomics of the next years. This article is part of a Special Issue entitled: 20years of Proteomics in memory of Viatliano Pallini. Guest Editors: Luca Bini, Juan J. Calvete, Natacha Turck, Denis Hochstrasser and Jean-Charles Sanchez. PMID:24681132

  15. High-Throughput Proteomics

    NASA Astrophysics Data System (ADS)

    Zhang, Zhaorui; Wu, Si; Stenoien, David L.; Paša-Tolić, Ljiljana

    2014-06-01

    Mass spectrometry (MS)-based high-throughput proteomics is the core technique for large-scale protein characterization. Due to the extreme complexity of proteomes, sophisticated separation techniques and advanced MS instrumentation have been developed to extend coverage and enhance dynamic range and sensitivity. In this review, we discuss the separation and prefractionation techniques applied for large-scale analysis in both bottom-up (i.e., peptide-level) and top-down (i.e., protein-level) proteomics. Different approaches for quantifying peptides or intact proteins, including label-free and stable-isotope-labeling strategies, are also discussed. In addition, we present a brief overview of different types of mass analyzers and fragmentation techniques as well as selected emerging techniques.

  16. Proteomics: capacity versus utility.

    PubMed

    Harry, J L; Wilkins, M R; Herbert, B R; Packer, N H; Gooley, A A; Williams, K L

    2000-04-01

    Until recently scientists studied genes or proteins one at a time. With improvements in technology, new tools have become available to study the complex interactions that occur in biological systems. Global studies are required to do this, and these will involve genomic and proteomic approaches. High-throughput methods are necessary in each case because the number of genes and proteins in even the simplest of organisms are immense. In the developmental phase of genomics, the emphasis was on the generation and assembly of large amounts of nucleic acid sequence data. Proteomics is currently in a phase of technological development and establishment, and demonstrating the capacity for high throughput is a major challenge. However, funding bodies (both in the public and private sector) are increasingly focused on the usefulness of this capacity. Here we review the current state of proteome research in terms of capacity and utility.

  17. Biophotonics applied to proteomics.

    PubMed

    Faupel, Michel; Bonenfant, Débora; Schindler, Patrick; Bertrand, Eric; Mueller, Dieter; Stoeckli, Markus; Bitsch, Francis; Rohner, Tatiana; Staab, Dieter; Van Oostrum, Jan

    2007-01-01

    Since the completion of the human genome sequencing, our understanding of gene and protein function and their involvement in physiopathological states has increased dramatically, partly due to technological developments in photonics. Photonics is a very active area where new developments occur on a weekly basis, while established tools are adapted to fulfill the needs of other disciplines like genomics and proteomics. Biophotonics emerged at the interface of photonics and biology as a very straightforward and efficient approach to observe and manipulate living systems. In this chapter, we review the current applications of photonics and imaging to proteomics from 2D gels analysis to molecular imaging.

  18. In-depth Characterization of the Cerebrospinal Fluid (CSF) Proteome Displayed Through the CSF Proteome Resource (CSF-PR)*

    PubMed Central

    Guldbrandsen, Astrid; Vethe, Heidrun; Farag, Yehia; Oveland, Eystein; Garberg, Hilde; Berle, Magnus; Myhr, Kjell-Morten; Opsahl, Jill A.; Barsnes, Harald; Berven, Frode S.

    2014-01-01

    In this study, the human cerebrospinal fluid (CSF) proteome was mapped using three different strategies prior to Orbitrap LC-MS/MS analysis: SDS-PAGE and mixed mode reversed phase-anion exchange for mapping the global CSF proteome, and hydrazide-based glycopeptide capture for mapping glycopeptides. A maximal protein set of 3081 proteins (28,811 peptide sequences) was identified, of which 520 were identified as glycoproteins from the glycopeptide enrichment strategy, including 1121 glycopeptides and their glycosylation sites. To our knowledge, this is the largest number of identified proteins and glycopeptides reported for CSF, including 417 glycosylation sites not previously reported. From parallel plasma samples, we identified 1050 proteins (9739 peptide sequences). An overlap of 877 proteins was found between the two body fluids, whereas 2204 proteins were identified only in CSF and 173 only in plasma. All mapping results are freely available via the new CSF Proteome Resource (http://probe.uib.no/csf-pr), which can be used to navigate the CSF proteome and help guide the selection of signature peptides in targeted quantitative proteomics. PMID:25038066

  19. Improved recovery and identification of membrane proteins from rat hepatic cells using a centrifugal proteomic reactor.

    PubMed

    Zhou, Hu; Wang, Fangjun; Wang, Yuwei; Ning, Zhibin; Hou, Weimin; Wright, Theodore G; Sundaram, Meenakshi; Zhong, Shumei; Yao, Zemin; Figeys, Daniel

    2011-10-01

    Despite their importance in many biological processes, membrane proteins are underrepresented in proteomic analysis because of their poor solubility (hydrophobicity) and often low abundance. We describe a novel approach for the identification of plasma membrane proteins and intracellular microsomal proteins that combines membrane fractionation, a centrifugal proteomic reactor for streamlined protein extraction, protein digestion and fractionation by centrifugation, and high performance liquid chromatography-electrospray ionization-tandem MS. The performance of this approach was illustrated for the study of the proteome of ER and Golgi microsomal membranes in rat hepatic cells. The centrifugal proteomic reactor identified 945 plasma membrane proteins and 955 microsomal membrane proteins, of which 63 and 47% were predicted as bona fide membrane proteins, respectively. Among these proteins, >800 proteins were undetectable by the conventional in-gel digestion approach. The majority of the membrane proteins only identified by the centrifugal proteomic reactor were proteins with ≥ 2 transmembrane segments or proteins with high molecular mass (e.g. >150 kDa) and hydrophobicity. The improved proteomic reactor allowed the detection of a group of endocytic and/or signaling receptor proteins on the plasma membrane, as well as apolipoproteins and glycerolipid synthesis enzymes that play a role in the assembly and secretion of apolipoprotein B100-containing very low density lipoproteins. Thus, the centrifugal proteomic reactor offers a new analytical tool for structure and function studies of membrane proteins involved in lipid and lipoprotein metabolism.

  20. Quantitative proteomic survey of endoplasmic reticulum in mouse liver.

    PubMed

    Song, Yanping; Jiang, Ying; Ying, Wantao; Gong, Yan; Yan, Yujuan; Yang, Dong; Ma, Jie; Xue, Xiaofang; Zhong, Fan; Wu, Songfeng; Hao, Yunwei; Sun, Aihua; Li, Tao; Sun, Wei; Wei, Handong; Zhu, Yunping; Qian, Xiaohong; He, Fuchu

    2010-03-01

    To gain a better understanding of the critical function of the endoplasmic reticulum (ER) in liver, we carried out a proteomic survey of mouse liver ER. The ER proteome was profiled with a new three-dimensional, gel-based strategy. From 6152 and 6935 MS spectra, 903 and 1042 proteins were identified with at least two peptides matches at 95% confidence in the rough (r) and smooth (s) ER, respectively. Comparison of the rER and sER proteomes showed that calcium-binding proteins are significantly enriched in the sER suggesting that the ion-binding function of the ER is compartmentalized. Comparison of the rat and mouse ER proteomes showed that 662 proteins were common to both, comprising 53.5% and 49.3% of those proteomes, respectively. We proposed that these proteins were stably expressed proteins that were essential for the maintenance of ER function. GO annotation with a hypergeometric model proved this hypothesis. Unexpectedly, 210 unknown proteins and some proteins previously reported to occur in the cytosol were highly enriched in the ER. This study provides a reference map for the ER proteome of liver. Identification of new ER proteins will enhance our current understanding of the ER and also suggest new functions for this organelle.

  1. MitoMiner: a data warehouse for mitochondrial proteomics data.

    PubMed

    Smith, Anthony C; Blackshaw, James A; Robinson, Alan J

    2012-01-01

    MitoMiner (http://mitominer.mrc-mbu.cam.ac.uk/) is a data warehouse for the storage and analysis of mitochondrial proteomics data gathered from publications of mass spectrometry and green fluorescent protein tagging studies. In MitoMiner, these data are integrated with data from UniProt, Gene Ontology, Online Mendelian Inheritance in Man, HomoloGene, Kyoto Encyclopaedia of Genes and Genomes and PubMed. The latest release of MitoMiner stores proteomics data sets from 46 studies covering 11 different species from eumetazoa, viridiplantae, fungi and protista. MitoMiner is implemented by using the open source InterMine data warehouse system, which provides a user interface allowing users to upload data for analysis, personal accounts to store queries and results and enables queries of any data in the data model. MitoMiner also provides lists of proteins for use in analyses, including the new MitoMiner mitochondrial proteome reference sets that specify proteins with substantial experimental evidence for mitochondrial localization. As further mitochondrial proteomics data sets from normal and diseased tissue are published, MitoMiner can be used to characterize the variability of the mitochondrial proteome between tissues and investigate how changes in the proteome may contribute to mitochondrial dysfunction and mitochondrial-associated diseases such as cancer, neurodegenerative diseases, obesity, diabetes, heart failure and the ageing process.

  2. CAPER: a chromosome-assembled human proteome browsER.

    PubMed

    Guo, Feifei; Wang, Dan; Liu, Zhongyang; Lu, Liang; Zhang, Wei; Sun, Haiyan; Zhang, Hongxing; Ma, Jie; Wu, Songfeng; Li, Ning; Jiang, Ying; Zhu, Weimin; Qin, Jun; Xu, Ping; Li, Dong; He, Fuchu

    2013-01-01

    High-throughput mass spectrometry and antibody-based experiments have begun to produce a large amount of proteomic data sets. Chromosome-based visualization of these data sets and their annotations can help effectively integrate, organize, and analyze them. Therefore, we developed a web-based, user-friendly Chromosome-Assembled human Proteome browsER (CAPER). To display proteomic data sets and related annotations comprehensively, CAPER employs two distinct visualization strategies: track-view for the sequence/site information and the correspondence between proteome, transcriptome, genome, and chromosome and heatmap-view for the qualitative and quantitative functional annotations. CAPER supports data browsing at multiple scales through Google Map-like smooth navigation, zooming, and positioning with chromosomes as the reference coordinate. Both track-view and heatmap-view can mutually switch, providing a high-quality user interface. Taken together, CAPER will greatly facilitate the complete annotation and functional interpretation of the human genome by proteomic approaches, thereby making a significant contribution to the Chromosome-Centric Human Proteome Project and even the human physiology/pathology research. CAPER can be accessed at http://www.bprc.ac.cn/CAPE .

  3. Xylem sap proteomics.

    PubMed

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

    2014-01-01

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

  4. “Seed Proteomics"

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteomic analysis of seeds encounters some specific problems that do not impinge on analyses of other plant cells, tissues, or organs. There are anatomic considerations. Seeds comprise the seed coat, the storage organ(s), and the embryonic axis. Are these to be studied individually or as a compo...

  5. Proteomics of extremophiles.

    PubMed

    Burg, Dominic; Ng, Charmaine; Ting, Lily; Cavicchioli, Ricardo

    2011-08-01

    Functional genomic approaches, such as proteomics, greatly enhance the value of genome sequences by providing a global level assessment of which genes are expressed, when genes are expressed and at what cellular levels gene products are synthesized. With over 1000 complete genome sequences of different microorganisms available, and DNA sequencing for environmental samples (metagenomes) producing vast amounts of gene sequence data, there is a real opportunity and a clear need to generate associated functional genomic data to learn about the source microorganisms. In contrast to the technological advances that have led to the accelerated rate and ease at which DNA sequence data can be generated, mass spectrometry based proteomics remains a technically sophisticated and exacting science. In recognition of the need to make proteomics more accessible to a growing number of environmental microbiologists so that the 'functional genomics gap' may be bridged, this review strives to demystify proteomic technologies and describe ways in which they have been applied, and more importantly, can be applied to study the physiology and ecology of extremophiles.

  6. Genomes to Proteomes

    SciTech Connect

    Panisko, Ellen A.; Grigoriev, Igor; Daly, Don S.; Webb-Robertson, Bobbie-Jo; Baker, Scott E.

    2009-03-01

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

  7. A Normative Study of the Synovial Fluid Proteome from Healthy Porcine Knee Joints

    PubMed Central

    2015-01-01

    Synovial fluid in an articulating joint contains proteins derived from the blood plasma and proteins that are produced by cells within the joint tissues, such as synovium, cartilage, ligament, and meniscus. The proteome composition of healthy synovial fluid and the cellular origins of many synovial fluid components are not fully understood. Here, we present a normative proteomics study using porcine synovial fluid. Using our optimized method, we identified 267 proteins with high confidence in healthy synovial fluid. We also evaluated mRNA expression data from tissues that can contribute to the synovial fluid proteome, including synovium, cartilage, blood, and liver, to better estimate the relative contributions from these sources to specific synovial fluid components. We identified 113 proteins in healthy synovial fluid that appear to be primarily derived from plasma transudates, 37 proteins primarily derived from synovium, and 11 proteins primarily derived from cartilage. Finally, we compared the identified synovial fluid proteome to the proteome of human plasma, and we found that the two body fluids share many similarities, underlining the detected plasma derived nature of many synovial fluid components. Knowing the synovial fluid proteome of a healthy joint will help to identify mechanisms that cause joint disease and pathways involved in disease progression. PMID:25160569

  8. A "proteomic ruler" for protein copy number and concentration estimation without spike-in standards.

    PubMed

    Wiśniewski, Jacek R; Hein, Marco Y; Cox, Jürgen; Mann, Matthias

    2014-12-01

    Absolute protein quantification using mass spectrometry (MS)-based proteomics delivers protein concentrations or copy numbers per cell. Existing methodologies typically require a combination of isotope-labeled spike-in references, cell counting, and protein concentration measurements. Here we present a novel method that delivers similar quantitative results directly from deep eukaryotic proteome datasets without any additional experimental steps. We show that the MS signal of histones can be used as a "proteomic ruler" because it is proportional to the amount of DNA in the sample, which in turn depends on the number of cells. As a result, our proteomic ruler approach adds an absolute scale to the MS readout and allows estimation of the copy numbers of individual proteins per cell. We compare our protein quantifications with values derived via the use of stable isotope labeling by amino acids in cell culture and protein epitope signature tags in a method that combines spike-in protein fragment standards with precise isotope label quantification. The proteomic ruler approach yields quantitative readouts that are in remarkably good agreement with results from the precision method. We attribute this surprising result to the fact that the proteomic ruler approach omits error-prone steps such as cell counting or protein concentration measurements. The proteomic ruler approach is readily applicable to any deep eukaryotic proteome dataset-even in retrospective analysis-and we demonstrate its usefulness with a series of mouse organ proteomes.

  9. Parallel barcoding of antibodies for DNA-assisted proteomics.

    PubMed

    Dezfouli, Mahya; Vickovic, Sanja; Iglesias, Maria Jesus; Schwenk, Jochen M; Ahmadian, Afshin

    2014-11-01

    DNA-assisted proteomics technologies enable ultra-sensitive measurements in multiplex format using DNA-barcoded affinity reagents. Although numerous antibodies are available, nowadays targeting nearly the complete human proteome, the majority is not accessible at the quantity, concentration, or purity recommended for most bio-conjugation protocols. Here, we introduce a magnetic bead-assisted DNA-barcoding approach, applicable for several antibodies in parallel, as well as reducing required reagents quantities up to a thousand-fold. The success of DNA-barcoding and retained functionality of antibodies were demonstrated in sandwich immunoassays and standard quantitative Immuno-PCR assays. Specific DNA-barcoding of antibodies for multiplex applications was presented on suspension bead arrays with read-out on a massively parallel sequencing platform in a procedure denoted Immuno-Sequencing. Conclusively, human plasma samples were analyzed to indicate the functionality of barcoded antibodies in intended proteomics applications. PMID:25263329

  10. Biomarker Discovery in Neurodegenerative Diseases: A Proteomic Approach

    PubMed Central

    Shi, Min; Caudle, W. Michael; Zhang, Jing

    2010-01-01

    Biomarkers for neurodegenerative disorders are essential to facilitate disease diagnosis, ideally at early stages, monitor disease progression, and assess response to existing and future treatments. Application of proteomics to the human brain, cerebrospinal fluid and plasma has greatly hastened the unbiased and high-throughput searches for novel biomarkers. There are many steps critical to biomarker discovery, whether for neurodegenerative or other diseases, including sample preparation, protein/peptide separation and identification, as well as independent confirmation and validation. In this review we have summarized current proteomics technologies involved in discovery of biomarkers for neurodegenerative diseases, practical considerations and limitations of several major aspects, as well as the current status of candidate biomarkers revealed by proteomics for Alzheimer and Parkinson diseases. PMID:18938247

  11. The Human Eye Proteome Project: perspectives on an emerging proteome.

    PubMed

    Semba, Richard D; Enghild, Jan J; Venkatraman, Vidya; Dyrlund, Thomas F; Van Eyk, Jennifer E

    2013-08-01

    There are an estimated 285 million people with visual impairment worldwide, of whom 39 million are blind. The pathogenesis of many eye diseases remains poorly understood. The human eye is currently an emerging proteome that may provide key insight into the biological pathways of disease. We review proteomic investigations of the human eye and present a catalogue of 4842 nonredundant proteins identified in human eye tissues and biofluids to date. We highlight the need to identify new biomarkers for eye diseases using proteomics. Recent advances in proteomics do now allow the identification of hundreds to thousands of proteins in tissues and fluids, characterization of various PTMs and simultaneous quantification of multiple proteins. To facilitate proteomic studies of the eye, the Human Eye Proteome Project (HEPP) was organized in September 2012. The HEPP is one of the most recent components of the Biology/Disease-driven Human Proteome Project (B/D-HPP) whose overarching goal is to support the broad application of state-of-the-art measurements of proteins and proteomes by life scientists studying the molecular mechanisms of biological processes and human disease. The large repertoire of investigative proteomic tools has great potential to transform vision science and enhance understanding of physiology and disease processes that affect sight.

  12. Proteomic analysis of hyperadhesive Candida glabrata clinical isolates reveals a core wall proteome and differential incorporation of adhesins.

    PubMed

    Gómez-Molero, Emilia; de Boer, Albert D; Dekker, Henk L; Moreno-Martínez, Ana; Kraneveld, Eef A; Ichsan; Chauhan, Neeraj; Weig, Michael; de Soet, Johannes J; de Koster, Chris G; Bader, Oliver; de Groot, Piet W J

    2015-12-01

    Attachment to human host tissues or abiotic medical devices is a key step in the development of infections by Candida glabrata. The genome of this pathogenic yeast codes for a large number of adhesins, but proteomic work using reference strains has shown incorporation of only few adhesins in the cell wall. By making inventories of the wall proteomes of hyperadhesive clinical isolates and reference strain CBS138 using mass spectrometry, we describe the cell wall proteome of C. glabrata and tested the hypothesis that hyperadhesive isolates display differential incorporation of adhesins. Two clinical strains (PEU382 and PEU427) were selected, which both were hyperadhesive to polystyrene and showed high surface hydrophobicity. Cell wall proteome analysis under biofilm-forming conditions identified a core proteome of about 20 proteins present in all C. glabrata strains. In addition, 12 adhesin-like wall proteins were identified in the hyperadherent strains, including six novel adhesins (Awp8-13) of which only Awp12 was also present in CBS138. We conclude that the hyperadhesive capacity of these two clinical C. glabrata isolates is correlated with increased and differential incorporation of cell wall adhesins. Future studies should elucidate the role of the identified proteins in the establishment of C. glabrata infections. PMID:26546455

  13. Proteome research in food science.

    PubMed

    Pischetsrieder, Monika; Baeuerlein, Rainer

    2009-09-01

    The proteome is the totality of proteins present in a biological sample. In contrast to the static genome, the proteome is highly dynamic, influenced by the genome and many external factors, such as the state of development, tissue type, metabolic state, and various interactions. Thus, the proteome reflects very closely the biological (and chemical) processes occurring in a system. For proteome analysis, gel based and shotgun methods are most widely applied. Because of the potential to generate a systematic view of protein composition and biological as well as chemical interactions, the application of proteome analysis in food science is steadily growing. This tutorial review introduces several fields in food science, where proteomics has been successfully applied: analysis of food composition, safety assessment of genetically modified food, the search for marker proteins for food authentication, identification of food allergens, systematic analysis of the physiological activity of food, analysis of the effects of processing on food proteins and the improvement of food quality.

  14. Proteomics Discovery of Disease Biomarkers.

    PubMed

    Ahram, Mamoun; Petricoin, Emanuel F

    2008-01-01

    Recent technological developments in proteomics have shown promising initiatives in identifying novel biomarkers of various diseases. Such technologies are capable of investigating multiple samples and generating large amount of data end-points. Examples of two promising proteomics technologies are mass spectrometry, including an instrument based on surface enhanced laser desorption/ionization, and protein microarrays. Proteomics data must, however, undergo analytical processing using bioinformatics. Due to limitations in proteomics tools including shortcomings in bioinformatics analysis, predictive bioinformatics can be utilized as an alternative strategy prior to performing elaborate, high-throughput proteomics procedures. This review describes mass spectrometry, protein microarrays, and bioinformatics and their roles in biomarker discovery, and highlights the significance of integration between proteomics and bioinformatics.

  15. Fourteen years of plant proteomics reflected in Proteomics: moving from model species and 2DE-based approaches to orphan species and gel-free platforms.

    PubMed

    Jorrín-Novo, Jesus V; Pascual, Jesus; Sánchez-Lucas, Rosa; Romero-Rodríguez, M Cristina; Rodríguez-Ortega, Manuel J; Lenz, Christof; Valledor, Luis

    2015-03-01

    In this article, the topic of plant proteomics is reviewed based on related papers published in the journal Proteomics since publication of the first issue in 2001. In total, around 300 original papers and 41 reviews published in Proteomics between 2000 and 2014 have been surveyed. Our main objective for this review is to help bridge the gap between plant biologists and proteomics technologists, two often very separate groups. Over the past years a number of reviews on plant proteomics have been published . To avoid repetition we have focused on more recent literature published after 2010, and have chosen to rather make continuous reference to older publications. The use of the latest proteomics techniques and their integration with other approaches in the "systems biology" direction are discussed more in detail. Finally we comment on the recent history, state of the art, and future directions of plant proteomics, using publications in Proteomics to illustrate the progress in the field. The review is organized into two major blocks, the first devoted to provide an overview of experimental systems (plants, plant organs, biological processes) and the second one to the methodology.

  16. Isotope labeled internal standards (ILIS) as a basis for quality control in clinical studies using plasma samples.

    PubMed

    Rezeli, Melinda; Végvári, Akos; Marko-Varga, György; Laurell, Thomas

    2010-04-18

    For clinical proteomic studies, the quality of the biofluid samples such as human blood plasma is extremely important. In this study we have investigated the stability of human plasma samples by spiking stable isotope-labeled peptides into the plasma and monitoring their degradation under different storage conditions. FPA-1, C4A and C3f were synthesized with isotopically labeled amino acids, and used as reference peptides. The mixture of internal calibrants was spiked into plasma at the starting point of investigation, mimicking the time of collection for future biobanking efforts, and their qualitative and quantitative changes were analyzed over time by using both MALDI-MS (LTQ Orbitrap XL) and nanoLC-ESI-MS (LTQ XL ETD). We have found that all three synthetic peptides were stable in plasma at -20 and -80 degrees C during the examined 2-month period. However, different proteolytic degradation profiles of the peptides were observed at room temperature. We anticipate that the use of these isotope-labeled peptides as internal standards (ILIS) provides a quality control for long-term storage and proteomic plasma analysis.

  17. Proteomics of blood and derived products: what's next?

    PubMed

    Prudent, Michel; Tissot, Jean-Daniel; Lion, Niels

    2011-12-01

    Proteomics has changed the way proteins are analyzed in living systems. This approach has been applied to blood products and protein profiling has evolved in parallel with the development of techniques. The identification of proteins belonging to red blood cell, platelets or plasma was achieved at the end of the last century. Then, the questions on the applications emerged. Hence, several studies have focused on problems related to blood banking and products, such as the aging of blood products, identification of biomarkers, related diseases and the protein-protein interactions. More recently, a mass spectrometry-based proteomics approach to quality control has been applied in order to offer solutions and improve the quality of blood products. The current challenge we face is developing a closer relationship between transfusion medicine and proteomics. In this article, these issues will be approached by focusing first on the proteome identification of blood products and then on the applications and future developments within the field of proteomics and blood products. PMID:22087657

  18. The Human Liver Proteome Project (HLPP) Workshop August 2008, Amsterdam, The Netherlands

    PubMed Central

    Beretta, Laura

    2015-01-01

    An HLPP workshop was held in conjunction with the 7th HUPO 2008 World Congress in Amsterdam, The Netherlands. The workshop was chaired by Laura Beretta (Fred Hutchinson Cancer Research Center (FHCRC)) and Xiaohong Qian (Beijing Proteome Research Center (BPRC)). The workshop was organized in three sessions: Session 1: The Human Proteome Project: the liver perspective, moderated by John Bergeron (McGill University); Session 2: The comparative analysis of the liver and plasma proteomes, moderated by Young-Ki Paik (Yonsei University) and Session 3: Opportunities for the study of liver diseases within the HLPP, moderated by Chantal Housset (INSERM, France). PMID:19862759

  19. Proteomics of the Lysosome

    PubMed Central

    Lübke, Torben; Lobel, Peter; Sleat, David

    2009-01-01

    Defects in lysosomal function have been associated with numerous monogenic human diseases typically classified as lysosomal storage diseases. However, there is increasing evidence that lysosomal proteins are also involved in more widespread human diseases including cancer and Alzheimer disease. Thus, there is a continuing interest in understanding the cellular functions of the lysosome and an emerging approach to this is the identification of its constituent proteins by proteomic analyses. To date, the mammalian lysosome has been shown to contain ~ 60 soluble luminal proteins and ~25 transmembrane proteins. However, recent proteomic studies based upon affinity purification of soluble components or subcellular fractionation to obtain both soluble and membrane components suggest that there may be many more of both classes of protein resident within this organelle than previously appreciated. Discovery of such proteins has important implications for understanding the function and the dynamics of the lysosome but can also lead the way towards the discovery of the genetic basis for human diseases of hitherto unknown etiology. Here, we describe current approaches to lysosomal proteomics and data interpretation and review the new lysosomal proteins that have recently emerged from such studies. PMID:18977398

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

    PubMed Central

    Meisrimler, Claudia-Nicole; Menckhoff, Ljiljana; Kukavica, Biljana M.; Lüthje, Sabine

    2015-01-01

    Legumes are important crop plants and pea (Pisum sativum L.) has been investigated as a model with respect to several physiological aspects. The sequencing of the pea genome has not been completed. Therefore, proteomic approaches are currently limited. Nevertheless, the increasing numbers of available EST-databases as well as the high homology of the pea and medicago genome (Medicago truncatula Gaertner) allow the successful identification of proteins. Due to the un-sequenced pea genome, pre-fractionation approaches have been used in pea proteomic surveys in the past. Aside from a number of selective proteome studies on crude extracts and the chloroplast, few studies have targeted other components such as the pea secretome, an important sub-proteome of interest due to its role in abiotic and biotic stress processes. The secretome itself can be further divided into different sub-proteomes (plasma membrane, apoplast, cell wall proteins). Cell fractionation in combination with different gel-electrophoresis, chromatography methods and protein identification by mass spectrometry are important partners to gain insight into pea sub-proteomes, post-translational modifications and protein functions. Overall, pea proteomics needs to link numerous existing physiological and biochemical data to gain further insight into adaptation processes, which play important roles in field applications. Future developments and directions in pea proteomics are discussed. PMID:26539198

  1. Circulating protein synthesis rates reveal skeletal muscle proteome dynamics

    PubMed Central

    Shankaran, Mahalakshmi; King, Chelsea L.; Angel, Thomas E.; Holmes, William E.; Li, Kelvin W.; Colangelo, Marc; Price, John C.; Turner, Scott M.; Bell, Christopher; Hamilton, Karyn L.; Miller, Benjamin F.; Hellerstein, Marc K.

    2015-01-01

    Here, we have described and validated a strategy for monitoring skeletal muscle protein synthesis rates in rodents and humans over days or weeks from blood samples. We based this approach on label incorporation into proteins that are synthesized specifically in skeletal muscle and escape into the circulation. Heavy water labeling combined with sensitive tandem mass spectrometric analysis allowed integrated synthesis rates of proteins in muscle tissue across the proteome to be measured over several weeks. Fractional synthesis rate (FSR) of plasma creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) in the blood, more than 90% of which is derived from skeletal muscle, correlated closely with FSR of CK-M, CA-3, and other proteins of various ontologies in skeletal muscle tissue in both rodents and humans. Protein synthesis rates across the muscle proteome generally changed in a coordinate manner in response to a sprint interval exercise training regimen in humans and to denervation or clenbuterol treatment in rodents. FSR of plasma CK-M and CA-3 revealed changes and interindividual differences in muscle tissue proteome dynamics. In human subjects, sprint interval training primarily stimulated synthesis of structural and glycolytic proteins. Together, our results indicate that this approach provides a virtual biopsy, sensitively revealing individualized changes in proteome-wide synthesis rates in skeletal muscle without a muscle biopsy. Accordingly, this approach has potential applications for the diagnosis, management, and treatment of muscle disorders. PMID:26657858

  2. Imaging proteomics for diagnosis, monitoring and prediction of Alzheimer's disease.

    PubMed

    Nazeri, Arash; Ganjgahi, Habib; Roostaei, Tina; Nichols, Thomas; Zarei, Mojtaba

    2014-11-15

    Proteomic and imaging markers have been widely studied as potential biomarkers for diagnosis, monitoring and prognosis of Alzheimer's disease. In this study, we used Alzheimer Disease Neuroimaging Initiative dataset and performed parallel independent component analysis on cross sectional and longitudinal proteomic and imaging data in order to identify the best proteomic model for diagnosis, monitoring and prediction of Alzheimer disease (AD). We used plasma proteins measurement and imaging data from AD and healthy controls (HC) at the baseline and 1 year follow-up. Group comparisons at baseline and changes over 1 year were calculated for proteomic and imaging data. The results were fed into parallel independent component analysis in order to identify proteins that were associated with structural brain changes cross sectionally and longitudinally. Regression model was used to find the best model that can discriminate AD from HC, monitor AD and to predict MCI converters from non-converters. We showed that five proteins are associated with structural brain changes in the brain. These proteins could discriminate AD from HC with 57% specificity and 89% sensitivity. Four proteins whose change over 1 year were associated with brain structural changes could discriminate AD from HC with sensitivity of 93%, and specificity of 92%. This model predicted MCI conversion to AD in 2 years with 94% accuracy. This model has the highest accuracy in prediction of MCI conversion to AD within the ADNI-1 dataset. This study shows that combination of selected plasma protein levels and MR imaging is a useful method in identifying potential biomarker.

  3. Proteome alterations in response to aristolochic acids in experimental animal model.

    PubMed

    Rucevic, Marijana; Rosenquist, Thomas; Breen, Lucas; Cao, Lulu; Clifton, James; Hixson, Douglas; Josic, Djuro

    2012-12-01

    Strong indications have been presented that dietary poisoning with aristolochic acids (AA) is responsible for Endemic Nephropathy (EN) and AA associated cancer of the upper urinary tract (UUTC). Our recent investigation showed drastic urinary proteome changes in AA treated mice. This study was designed to identify proteome changes associated with AA nephrotoxicity in experimental animal model. The DBA and C57BL mice, which differ in AA sensitivity, were exposed to AA for 4 days. The strategy for urinary, plasma and kidney tissue proteome study of AA exposed and control mice integrated gel-based and in-solution tryptic digestion combined with LC-ESI-MS/MS. To maximize proteome coverage, plasma fractionation scheme was developed and MS compatible sequential tissue extraction procedure was established. Proteomic analyses of urinary, plasma and kidney tissue tryptic digests resulted in identification of several cytoskeletal proteins, as well as proteins involved in kidney development and inflammatory response, that are differentially expressed in both AA exposed and control mice. These proteins are consistent with renal pathogenesis of endotoxicity and cancer. This proteomic strategy could be effectively translated for unbiased discovery of potential biomarkers for EN and associated UUTC in humans. At the same time, these results highlight the significance of AA exposure with EN. This article is part of a Special Issue entitled: Integrated omics.

  4. Accurate determination of chlorine, bromine, and iodine in sedimentary rock reference samples by radiochemical neutron activation analysis and a detailed comparison with inductively coupled plasma mass spectrometry literature data.

    PubMed

    Sekimoto, Shun; Ebihara, Mitsuru

    2013-07-01

    Trace amounts of three halogens (chlorine, bromine, and iodine) were determined using radiochemical neutron activation analysis (RNAA) for nine sedimentary rocks and three rhyolite samples. To obtain high-quality analytical data, the radiochemical procedure of RNAA was improved by lowering the background in gamma-ray spectrometry and completing the chemical procedure more rapidly than in conventional procedures. A comparison of the RNAA data of Br and I with corresponding inductively coupled plasma mass spectrometry (ICPMS) literature data revealed that the values obtained by ICPMS coupled with pyrohydrolysis preconcentration were systematically lower than the RNAA data for some reference samples, suggesting that the quantitative collection of Br and I cannot always be achieved by the pyrohydrolysis for some solid samples. The RNAA data of three halogens can classify sedimentary rock reference samples into two groups (the samples from inland water and those from seawater), implying the geochemical significance of halogen data.

  5. Accurate determination of chlorine, bromine, and iodine in sedimentary rock reference samples by radiochemical neutron activation analysis and a detailed comparison with inductively coupled plasma mass spectrometry literature data.

    PubMed

    Sekimoto, Shun; Ebihara, Mitsuru

    2013-07-01

    Trace amounts of three halogens (chlorine, bromine, and iodine) were determined using radiochemical neutron activation analysis (RNAA) for nine sedimentary rocks and three rhyolite samples. To obtain high-quality analytical data, the radiochemical procedure of RNAA was improved by lowering the background in gamma-ray spectrometry and completing the chemical procedure more rapidly than in conventional procedures. A comparison of the RNAA data of Br and I with corresponding inductively coupled plasma mass spectrometry (ICPMS) literature data revealed that the values obtained by ICPMS coupled with pyrohydrolysis preconcentration were systematically lower than the RNAA data for some reference samples, suggesting that the quantitative collection of Br and I cannot always be achieved by the pyrohydrolysis for some solid samples. The RNAA data of three halogens can classify sedimentary rock reference samples into two groups (the samples from inland water and those from seawater), implying the geochemical significance of halogen data. PMID:23710630

  6. Overflow metabolism in E. coli results from efficient proteome allocation

    PubMed Central

    Okano, Hiroyuki; Zhang, Zhongge; Shen, Yang; Williamson, James R.; Hwa, Terence

    2015-01-01

    Overflow metabolism refers to the seemingly wasteful strategy in which cells use fermentation instead of the more efficient respiration to generate energy, despite the availability of oxygen. Known as Warburg effect in the context of cancer growth, this phenomenon occurs ubiquitously for fast growing cells, including bacteria, fungi, and mammalian cells, but its origin has remained mysterious despite decades of research. Here we study metabolic overflow in E. coli and show that it is a global physiological response used to cope with changing proteomic demands of energy biogenesis and biomass synthesis under different growth conditions. A simple model of proteomic resource allocation can quantitatively account for all of the observed behaviors and accurately predict responses to novel perturbations. The key hypothesis of the model, that the proteome cost of energy biogenesis by respiration exceeds that by fermentation, is quantitatively confirmed by direct measurement of protein abundances via quantitative mass spectrometry. PMID:26632588

  7. Overflow metabolism in Escherichia coli results from efficient proteome allocation.

    PubMed

    Basan, Markus; Hui, Sheng; Okano, Hiroyuki; Zhang, Zhongge; Shen, Yang; Williamson, James R; Hwa, Terence

    2015-12-01

    Overflow metabolism refers to the seemingly wasteful strategy in which cells use fermentation instead of the more efficient respiration to generate energy, despite the availability of oxygen. Known as the Warburg effect in the context of cancer growth, this phenomenon occurs ubiquitously for fast-growing cells, including bacteria, fungi and mammalian cells, but its origin has remained unclear despite decades of research. Here we study metabolic overflow in Escherichia coli, and show that it is a global physiological response used to cope with changing proteomic demands of energy biogenesis and biomass synthesis under different growth conditions. A simple model of proteomic resource allocation can quantitatively account for all of the observed behaviours, and accurately predict responses to new perturbations. The key hypothesis of the model, that the proteome cost of energy biogenesis by respiration exceeds that by fermentation, is quantitatively confirmed by direct measurement of protein abundances via quantitative mass spectrometry.

  8. Proteomics Approaches Shed New Light on Traditional Iranian Medicine

    PubMed Central

    Movahhed, Mina; Poursaleh, Zohreh

    2016-01-01

    Background: Until now, Iranian traditional medicine (ITM) had been extensively based on Iranian philosophy in theoretical approach in diagnosis and treatment, with doubts on academic medicine. Nevertheless, the diagnosis of temperaments, herbal standardization, and quality control had been with the obscurity of functional molecules and their action mechanisms. Proteomics is a potent board to the mechanistic investigation of ITM and has been comprehensively applied profile drug-regulated proteins. In this review, we assessed the application of this modern molecular biological method in the identification of temperaments and drug targets of ITM. Methods: All available studies related to proteomics in traditional medicine, alternative and complementary medicine, including books, journals, and other references were studied and assessed. Results: The present review showed the phenotypes of the various temperaments in healthy individuals, that is to say, same proteins with different dynamic properties. Therefore, the usefulness of proteomics seems authoritative to understand the means by which the molecular pathways protected in ITM. This might be also the key clinical viewpoint on this new approach for enabling the integration of Iranian traditional medicine and modern biological science and technology, as well for upholding the internationalization of ITM. Conclusion: Proteomics, as a powerful tool for systems biology, is an essential research methodology for understanding the mechanisms of traditional medicine. Further investigation on the applications of advanced proteomics in temperaments, herbal standardization, and quality control in ITM is recommended. PMID:27516684

  9. Achievements and perspectives of top-down proteomics.

    PubMed

    Armirotti, Andrea; Damonte, Gianluca

    2010-10-01

    Over the last years, top-down (TD) MS has gained a remarkable space in proteomics, rapidly trespassing the limit between a promising approach and a solid, established technique. Several research groups worldwide have implemented TD analysis in their routine work on proteomics, deriving structural information on proteins with the level of accuracy that is impossible to achieve with classical bottom-up approaches. Complete maps of PTMs and assessment of single aminoacid polymorphisms are only a few of the results that can be obtained with this technique. Despite some existing technical and economical limitations, TD analysis is at present the most powerful instrument for MS-based proteomics and its implementation in routine workflow is a rapidly approaching turning point in proteomics. In this review article, the state-of-the-art of TD approach is described along with its major advantages and drawbacks and the most recent trends in TD analysis are discussed. References for all the covered topics are reported in the text, with the aim to support both newcomers and mass spectrometrists already introduced to TD proteomics.

  10. Proteome Studies of Filamentous Fungi

    SciTech Connect

    Baker, Scott E.; Panisko, Ellen A.

    2011-04-20

    The continued fast pace of fungal genome sequence generation has enabled proteomic analysis of a wide breadth of organisms that span the breadth of the Kingdom Fungi. There is some phylogenetic bias to the current catalog of fungi with reasonable DNA sequence databases (genomic or EST) that could be analyzed at a global proteomic level. However, the rapid development of next generation sequencing platforms has lowered the cost of genome sequencing such that in the near future, having a genome sequence will no longer be a time or cost bottleneck for downstream proteomic (and transcriptomic) analyses. High throughput, non-gel based proteomics offers a snapshot of proteins present in a given sample at a single point in time. There are a number of different variations on the general method and technologies for identifying peptides in a given sample. We present a method that can serve as a “baseline” for proteomic studies of fungi.

  11. Asymmetric Proteome Equalization of the Skeletal Muscle Proteome Using a Combinatorial Hexapeptide Library

    PubMed Central

    Rivers, Jenny; Hughes, Chris; McKenna, Thérèse; Woolerton, Yvonne; Vissers, Johannes P. C.; Langridge, James I.; Beynon, Robert J.

    2011-01-01

    Immobilized combinatorial peptide libraries have been advocated as a strategy for equalization of the dynamic range of a typical proteome. The technology has been applied predominantly to blood plasma and other biological fluids such as urine, but has not been used extensively to address the issue of dynamic range in tissue samples. Here, we have applied the combinatorial library approach to the equalization of a tissue where there is also a dramatic asymmetry in the range of abundances of proteins; namely, the soluble fraction of skeletal muscle. We have applied QconCAT and label-free methodology to the quantification of the proteins that bind to the beads as the loading is progressively increased. Although some equalization is achieved, and the most abundant proteins no longer dominate the proteome analysis, at high protein loadings a new asymmetry of protein expression is reached, consistent with the formation of complex assembles of heat shock proteins, cytoskeletal elements and other proteins on the beads. Loading at different ionic strength values leads to capture of different subpopulations of proteins, but does not completely eliminate the bias in protein accumulation. These assemblies may impair the broader utility of combinatorial library approaches to the equalization of tissue proteomes. However, the asymmetry in equalization is manifest at either low and high ionic strength values but manipulation of the solvent conditions may extend the capacity of the method. PMID:22205978

  12. Proteomics research in India: an update.

    PubMed

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

    2015-09-01

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

  13. Proteomics research in India: an update.

    PubMed

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

    2015-09-01

    After a successful completion of the Human Genome Project, deciphering the mystery surrounding the human proteome posed a major challenge. Despite not being largely involved in the Human Genome Project, the Indian scientific community contributed towards proteomic research along with the global community. Currently, more than 76 research/academic institutes and nearly 145 research labs are involved in core proteomic research across India. The Indian researchers have been major contributors in drafting the "human proteome map" along with international efforts. In addition to this, virtual proteomics labs, proteomics courses and remote triggered proteomics labs have helped to overcome the limitations of proteomics education posed due to expensive lab infrastructure. The establishment of Proteomics Society, India (PSI) has created a platform for the Indian proteomic researchers to share ideas, research collaborations and conduct annual conferences and workshops. Indian proteomic research is really moving forward with the global proteomics community in a quest to solve the mysteries of proteomics. A draft map of the human proteome enhances the enthusiasm among intellectuals to promote proteomic research in India to the world.This article is part of a Special Issue entitled: Proteomics in India. PMID:25868663

  14. proBAMsuite, a Bioinformatics Framework for Genome-Based Representation and Analysis of Proteomics Data*

    PubMed Central

    Wang, Xiaojing; Slebos, Robbert J. C.; Chambers, Matthew C.; Tabb, David L.; Liebler, Daniel C.; Zhang, Bing

    2016-01-01

    To facilitate genome-based representation and analysis of proteomics data, we developed a new bioinformatics framework, proBAMsuite, in which a central component is the protein BAM (proBAM) file format for organizing peptide spectrum matches (PSMs)1 within the context of the genome. proBAMsuite also includes two R packages, proBAMr and proBAMtools, for generating and analyzing proBAM files, respectively. Applying proBAMsuite to three recently published proteomics datasets, we demonstrated its utility in facilitating efficient genome-based sharing, interpretation, and integration of proteomics data. First, the interpretation of proteomics data is significantly enhanced with the rich genomic annotation information. Second, PSMs can be easily reannotated using user-specified gene annotation schemes and assembled into both protein and gene identifications. Third, using the genome as a common reference, proBAMsuite facilitates seamless proteomics and proteogenomics data integration. Finally, proBAM files can be readily visualized in genome browsers and thus bring proteomics data analysis to a general audience beyond the proteomics community. Results from this study establish proBAMsuite as a useful bioinformatics framework for proteomics and proteogenomics research. PMID:26657539

  15. Proteomics, oxidative stress and male infertility.

    PubMed

    Agarwal, Ashok; Durairajanayagam, Damayanthi; Halabi, Jacques; Peng, Jason; Vazquez-Levin, Monica

    2014-07-01

    Oxidative stress has been established as one of the main causes of male infertility and has been implicated in many diseases associated with infertile men. It results from high concentrations of free radicals and suppressed antioxidant potential, which may alter protein expression in seminal plasma and/or spermatozoa. In recent years, proteomic analyses have been performed to characterize the protein profiles of seminal ejaculate from men with different clinical conditions, such as high oxidative stress. The aim of the present review is to summarize current findings on proteomic studies performed in men with high oxidative stress compared with those with physiological concentrations of free radicals, to better understand the aetiology of oxidative stress-induced male infertility. Each of these studies has suggested candidate biomarkers of oxidative stress, among them are DJ-1, PIP, lactotransferrin and peroxiredoxin. Changes in protein concentrations in seminal plasma samples with oxidative stress conditions were related to stress responses and to regulatory pathways, while alterations in sperm proteins were mostly associated to metabolic responses (carbohydrate metabolism) and stress responses. Future studies should include assessment of post-translational modifications in the spermatozoa as well as in seminal plasma proteomes of men diagnosed with idiopathic infertility. Oxidative stress, which occurs due to a state of imbalance between free radicals and antioxidants, has been implicated in most cases of male infertility. Cells that are in a state of oxidative stress are more likely to have altered protein expression. The aim of this review is to better understand the causes of oxidative stress-induced male infertility. To achieve this, we assessed proteomic studies performed on the seminal plasma and spermatozoa of men with high levels of oxidative stress due to various clinical conditions and compared them with men who had physiological concentrations of free

  16. Reference Assessment

    ERIC Educational Resources Information Center

    Bivens-Tatum, Wayne

    2006-01-01

    This article presents interesting articles that explore several different areas of reference assessment, including practical case studies and theoretical articles that address a range of issues such as librarian behavior, patron satisfaction, virtual reference, or evaluation design. They include: (1) "Evaluating the Quality of a Chat Service"…

  17. Reference Revolutions.

    ERIC Educational Resources Information Center

    Mason, Marilyn Gell

    1998-01-01

    Describes developments in Online Computer Library Center (OCLC) electronic reference services. Presents a background on networked cataloging and the initial implementation of reference services by OCLC. Discusses the introduction of OCLC FirstSearch service, which today offers access to over 65 databases, future developments in integrated…

  18. NASCAP programmer's reference manual

    NASA Technical Reports Server (NTRS)

    Mandell, M. J.; Stannard, P. R.; Katz, I.

    1993-01-01

    The NASA Charging Analyzer Program (NASCAP) is a computer program designed to model the electrostatic charging of complicated three-dimensional objects, both in a test tank and at geosynchronous altitudes. This document is a programmer's reference manual and user's guide. It is designed as a reference to experienced users of the code, as well as an introduction to its use for beginners. All of the many capabilities of NASCAP are covered in detail, together with examples of their use. These include the definition of objects, plasma environments, potential calculations, particle emission and detection simulations, and charging analysis.

  19. Biochemical and Proteomic Characterization of Alkaptonuric Chondrocytes

    PubMed Central

    Braconi, Daniela; Bernardini, Giulia; Bianchini, Claretta; Laschi, Marcella; Millucci, Lia; Amato, Loredana; Tinti, Laura; Serchi, Tommaso; Chellini, Federico; Spreafico, Adriano; Santucci, Annalisa

    2012-01-01

    Alkaptonuria (AKU) is a rare genetic disease associated with the accumulation of homogentisic acid (HGA) and its oxidized/polymerized products which leads to the deposition of melanin-like pigments (ochronosis) in connective tissues. Although numerous case reports have described ochronosis in joints, little is known on the molecular mechanisms leading to such a phenomenon. For this reason, we characterized biochemically chondrocytes isolated from the ochronotic cartilage of AKU patients. Based on the macroscopic appearance of the ochronotic cartilage, two sub-populations were identified: cells coming from the black portion of the cartilage were referred to as “black” AKU chondrocytes, while those coming from the white portion were referred to as “white” AKU chondrocytes. Notably, both AKU chondrocytic types were characterized by increased apoptosis, NO release, and levels of pro-inflammatory cytokines. Transmission electron microscopy also revealed that intracellular ochronotic pigment deposition was common to both “white” and “black” AKU cells. We then undertook a proteomic and redox-proteomic analysis of AKU chondrocytes which revealed profound alterations in the levels of proteins involved in cell defence, protein folding, and cell organization. An increased post-translational oxidation of proteins, which also involved high molecular weight protein aggregates, was found to be particularly relevant in “black” AKU chondrocytes. J. Cell. Physiol. 227: 3333–3343, 2012. © 2011 Wiley Periodicals, Inc. PMID:22213341

  20. Erythrocyte incorporation of iron by infants: iron bioavailability from a low-iron infant formula and an evaluation of the usefulness of correcting erythrocyte incorporation values, using a reference dose or plasma ferritin concentrations.

    PubMed

    Davidsson, L; Ziegler, E E; Kastenmayer, P; Hurrell, R F

    2000-12-01

    Bioavailability of iron (Fe) from a low-Fe infant formula was determined by erythrocyte incorporation of 58Fe 14 d after administration in ten healthy, non-Fe-deficient infants. Two feeding protocols were compared, with each infant acting as his/her own control. At 140 and 154 d of age, infants were fed 1000 g of 58Fe-labelled formula (1.44 mg total Fe/1000 g) as six feeds over 24 h (Protocol A) or as two feeds/day on three consecutive days (Protocol B). A water solution with 57Fe and ascorbic acid was given separately as a reference dose in both study protocols. Erythrocyte incorporation of 58Fe and 57Fe was determined by thermal ionisation mass spectrometry. Geometric mean 58Fe incorporation was 7.6% (range 3.3-13.5%) with Protocol A as compared to 10.6% (range 6.7-18.6%) with Protocol B (P = 0.05); paired t test. Inter-individual variability of 58Fe was not reduced by correcting for the incorporation of 57Fe from the reference dose, or by correcting for plasma ferritin concentration. Fractional erythrocyte incorporation of Fe from low-Fe infant formula was in the same range as our earlier published data on erythrocyte incorporation of Fe from human milk extrinsically labelled with 58Fe (Davidsson et al. 1994a). The methodological evaluations included in this study clearly indicate the importance of using standardised study protocols when evaluating Fe bioavailability in infants. Corrections of erythrocyte incorporation data based on plasma ferritin or erythrocyte incorporation of Fe from a reference dose were not found to be useful.

  1. Proteome complexity and the forces that drive proteome imbalance.

    PubMed

    Harper, J Wade; Bennett, Eric J

    2016-01-01

    The cellular proteome is a complex microcosm of structural and regulatory networks that requires continuous surveillance and modification to meet the dynamic needs of the cell. It is therefore crucial that the protein flux of the cell remains in balance to ensure proper cell function. Genetic alterations that range from chromosome imbalance to oncogene activation can affect the speed, fidelity and capacity of protein biogenesis and degradation systems, which often results in proteome imbalance. An improved understanding of the causes and consequences of proteome imbalance is helping to reveal how these systems can be targeted to treat diseases such as cancer. PMID:27629639

  2. Determination of rare earth elements in human hair and wheat flour reference materials by inductively coupled plasma mass spectrometry with dry ashing and microwave digestion

    NASA Astrophysics Data System (ADS)

    Ming, Yin; Bing, Li

    1998-09-01

    A method was developed for the determination of all rare earth elements (REEs) at sub ng g -1 levels in human hair (GBW 09101, SRM, Republic of China) and wheat flour (GBW 08503, SRM, Republic of China) by Inductively coupled plasma mass spectrometry (ICP-MS). The values obtained by dry ashing and microwave oven digestion procedures were compared with those obtained by traditional open vessel acid digestion method. The validity of the analytical procedure was examined by analyzing spiked samples and two vegetables (GBW 07603 and GBW 07605, SRMs, Republic of China). The results are satisfactory. The detection limits for 14 REEs ranged from 0.0039 to 0.0003 ng cm -3 in solution and the quantification limits ranged from 0.16 to 0.01 ng g -1 in solid sample. The precision for most REEs were less than 10% RSD.

  3. Rumen-protected methionine and lysine: effects on milk production and plasma amino acids of dairy cows with reference to metabolisable protein status.

    PubMed

    Awawdeh, Mofleh S

    2016-05-01

    Two experiments were conducted to study the effects of rumen-protected Met (RPM) alone or with rumen-protected Lys (RPL) on milk yield and plasma amino acids of dairy cows. In experiment 1, 24 multiparous Holstein cows (154 DIM) were assigned to one of 3 groups where each cow received 0 g/d of RPM and RPL (C), 30 g/d of RPM (M), or 30 g/d of RPM plus 25 g of RPL (ML). The study lasted for 8 weeks where milk yield and composition were determined weekly. Daily milk yield averaged 28·0, 27·8, and 29·7 kg/cow for the C, M, and ML groups, respectively. Dietary treatments had no effects (P ≥ 0·54) on milk contents of fat, lactose, solid non-fat or total solids. Milk protein content in the ML group was greater (P < 0·05) than the C and M groups. Plasma levels of all AA were not significantly (P ≥ 0·09) affected by supplemental RPL and/or RPM. In experiment 2, 30 multiparous Holstein cows (100 DIM) were assigned to one of 3 groups where each cow received 0 g/d of RPM and RPL (C), 50 g/d of RPM (M), or 50 g/d of RPM plus 25 g/d of RPL (ML). The study lasted for 5 weeks. Cows in the M (30·5 kg) and ML (31·4 kg) groups produced (P < 0·05) more milk than those of the C group (29·1 kg). Under conditions of this study, RPM plus RPL improved milk yield and protein contents of dairy cows and was better than supplying RPM alone. Response in milk yield to RPM and RPL was affected by the MP status of cows which deserves further investigation.

  4. The seed nuclear proteome

    PubMed Central

    Repetto, Ombretta; Rogniaux, Hélène; Larré, Colette; Thompson, Richard; Gallardo, Karine

    2012-01-01

    Understanding the regulatory networks coordinating seed development will help to manipulate seed traits, such as protein content and seed weight, in order to increase yield and seed nutritional value of important food crops, such as legumes. Because of the cardinal role of the nucleus in gene expression, sub-proteome analyses of nuclei from developing seeds were conducted, taking advantage of the sequences available for model species. In this review, we discuss the strategies used to separate and identify the nuclear proteins at a stage when the seed is preparing for reserve accumulation. We present how these data provide an insight into the complexity and distinctive features of the seed nuclear proteome. We discuss the presence of chromatin-modifying enzymes and proteins that have roles in RNA-directed DNA methylation and which may be involved in modifying genome architecture in preparation for seed filling. Specific features of the seed nuclei at the transition between the stage of cell divisions and that of cell expansion and reserve deposition are described here which may help to manipulate seed quality traits, such as seed weight. PMID:23267364

  5. A new approach for calibration of laser ablation inductively coupled plasma mass spectrometry using thin layers of spiked agarose gels as references.

    PubMed

    Stärk, H-J; Wennrich, Rainer

    2011-02-01

    Calibration of analytical methods using laser ablation for sample introduction is often problematic. The availability of matrix-adapted standard materials is a crucial factor in the analysis of biological samples in particular. In this work a method for preparation of thin-film references for LA-ICP-MS is presented which is inexpensive, relatively simple and generally practicable. Aqueous solutions of agarose spiked with defined amounts of the analytes were cast on a carrier and then dried. When the thin-film references were characterized the average thickness of the films was 0.03 mm in the centre of the film and the relative standard deviation was 8%. Nebulization ICP-MS analysis after acid digestion of the agarose film was used to investigate the effectiveness of the spiking procedure. Recovery of the spiked elements was frequently in the range 90-110% (for rare earth elements 97-102%). Laser ablation ICP-MS analysis was used to investigate the distribution of the spiked elements in the film. When the laser was scanned across the gel the measured intensities were not constant, but had a peak-shaped profile with a flat top. Use of this flat-top region for analytical purposes, after its characterization by laser ablation ICP-MS, is proposed. Analysis of cell cultures was carried out by direct laser ablation-ICP-MS with the calibration method described. The results were in accordance with values previously achieved by nebulization ICP-MS.

  6. Ready Reference.

    ERIC Educational Resources Information Center

    Koltay, Emery

    1999-01-01

    Includes the following ready reference information: "Publishers' Toll-Free Telephone Numbers"; "How to Obtain an ISBN (International Standard Book Number)"; "How to Obtain an ISSN (International Standard Serial Number)"; and "How to Obtain an SAN (Standard Address Number)". (AEF)

  7. Immunocapture strategies in translational proteomics

    PubMed Central

    Fredolini, Claudia; Byström, Sanna; Pin, Elisa; Edfors, Fredrik; Tamburro, Davide; Iglesias, Maria Jesus; Häggmark, Anna; Hong, Mun-Gwan; Uhlen, Mathias; Nilsson, Peter; Schwenk, Jochen M

    2016-01-01

    Aiming at clinical studies of human diseases, antibody-assisted assays have been applied to biomarker discovery and toward a streamlined translation from patient profiling to assays supporting personalized treatments. In recent years, integrated strategies to couple and combine antibodies with mass spectrometry-based proteomic efforts have emerged, allowing for novel possibilities in basic and clinical research. Described in this review are some of the field’s current and emerging immunocapture approaches from an affinity proteomics perspective. Discussed are some of their advantages, pitfalls and opportunities for the next phase in clinical and translational proteomics. PMID:26558424

  8. Ovarian Cancer Proteomic, Phosphoproteomic, and Glycoproteomic Data Released - Office of Cancer Clinical Proteomics Research

    Cancer.gov

    National Cancer Institute (NCI) Clinical Proteomic Tumor Analysis Consortium (CPTAC) scientists have just released a comprehensive dataset of the proteomic analysis of high grade serous ovarian tumor samples,

  9. A guide to the Proteomics Identifications Database proteomics data repository.

    PubMed

    Vizcaíno, Juan Antonio; Côté, Richard; Reisinger, Florian; Foster, Joseph M; Mueller, Michael; Rameseder, Jonathan; Hermjakob, Henning; Martens, Lennart

    2009-09-01

    The Proteomics Identifications Database (PRIDE, www.ebi.ac.uk/pride) is one of the main repositories of MS derived proteomics data. Here, we point out the main functionalities of PRIDE both as a submission repository and as a source for proteomics data. We describe the main features for data retrieval and visualization available through the PRIDE web and BioMart interfaces. We also highlight the mechanism by which tailored queries in the BioMart can join PRIDE to other resources such as Reactome, Ensembl or UniProt to execute extremely powerful across-domain queries. We then present the latest improvements in the PRIDE submission process, using the new easy-to-use, platform-independent graphical user interface submission tool PRIDE Converter. Finally, we speak about future plans and the role of PRIDE in the ProteomExchange consortium.

  10. Redox Proteomics in Human Biofluids: Sample Preparation, Separation and Immunochemical Tagging for Analysis of Protein Oxidation.

    PubMed

    Di Domenico, Fabio; Perluigi, Marzia; Butterfield, D Allan

    2016-01-01

    Proteomics offers the simultaneous detection of a large number of proteins in a single experiment and can provide important information regarding crucial aspects of specific proteins, particularly post-translational modifications (PTMs). Investigations of oxidative PTMs are currently performed using focused redox proteomics techniques, which rely on gel electrophoresis separations of intact proteins with the final detection of oxidative PTMs being performed by mass spectrometry (MS) analysis. The application of this technique to human biofluids is being subject of increasing investigation and is expected to provide new insights on the oxidative status of the peripheral proteome in neurological diseases such as Alzheimer's disease, towards purposes of early diagnosis and prognosis. This chapter describes all the experimental steps to perform redox proteomics analysis of cerebrospinal fluid and plasma/serum samples.

  11. Proteomics in farm animals models of human diseases.

    PubMed

    Ceciliani, Fabrizio; Restelli, Laura; Lecchi, Cristina

    2014-10-01

    The need to provide in vivo complex environments to understand human diseases strongly relies on the use of animal models, which traditionally include small rodents and rabbits. It is becoming increasingly evident that the few species utilised to date cannot be regarded as universal. There is a great need for new animal species that are naturally endowed with specific features relevant to human diseases. Farm animals, including pigs, cows, sheep and horses, represent a valid alternative to commonly utilised rodent models. There is an ample scope for the application of proteomic techniques in farm animals, and the establishment of several proteomic maps of plasma and tissue has clearly demonstrated that farm animals provide a disease environment that closely resembles that of human diseases. The present review offers a snapshot of how proteomic techniques have been applied to farm animals to improve their use as biomedical models. Focus will be on specific topics of biomedical research in which farm animal models have been characterised through the application of proteomic techniques.

  12. Proteomics of early zebrafish embryos

    PubMed Central

    Link, Vinzenz; Shevchenko, Andrej; Heisenberg, Carl-Philipp

    2006-01-01

    Background Zebrafish (D. rerio) has become a powerful and widely used model system for the analysis of vertebrate embryogenesis and organ development. While genetic methods are readily available in zebrafish, protocols for two dimensional (2D) gel electrophoresis and proteomics have yet to be developed. Results As a prerequisite to carry out proteomic experiments with early zebrafish embryos, we developed a method to efficiently remove the yolk from large batches of embryos. This method enabled high resolution 2D gel electrophoresis and improved Western blotting considerably. Here, we provide detailed protocols for proteomics in zebrafish from sample preparation to mass spectrometry (MS), including a comparison of databases for MS identification of zebrafish proteins. Conclusion The provided protocols for proteomic analysis of early embryos enable research to be taken in novel directions in embryogenesis. PMID:16412219

  13. Proteomics in Rheumatoid Arthritis Research

    PubMed Central

    Park, Yune-Jung; Chung, Min Kyung; Hwang, Daehee

    2015-01-01

    Although rheumatoid arthritis (RA) is the most common chronic inflammatory autoimmune disease, diagnosis of RA is currently based on clinical manifestations, and there is no simple, practical assessment tool in the clinical field to assess disease activity and severity. Recently, there has been increasing interest in the discovery of new diagnostic RA biomarkers that can assist in evaluating disease activity, severity, and treatment response. Proteomics, the large-scale study of the proteome, has emerged as a powerful technique for protein identification and characterization. For the past 10 years, proteomic techniques have been applied to different biological samples (synovial tissue/fluid, blood, and urine) from RA patients and experimental animal models. In this review, we summarize the current state of the application of proteomics in RA and its importance in identifying biomarkers and treatment targets. PMID:26330803

  14. Proteomics: Technology Development and Applications

    PubMed Central

    Jayaraman, Arul

    2009-01-01

    Technology development in and the application of proteomics are emerging areas among the chemical engineers and others who presented at the 2008 American Institute of Chemical Engineers (AIChE) Annual Meeting. Overall, the centennial meeting offered a broad current perspective on the discipline of chemical engineering as it enters its second century. Biomedical and biochemical engineering continue to grow as important facets of the discipline. Within these, the value and applicability of proteomics were demonstrated in a number of interesting presentations. This year, as in the recent past, the AIChE Annual meeting was held in conjunction with the American Electrophoresis Society (AES) Annual Meeting. AES presenters offered further academic and industrial viewpoints on the still-developing role of proteomics and proteomic technologies in biological and clinical analyses. PMID:19210124

  15. Proteomics of Plant Pathogenic Fungi

    PubMed Central

    González-Fernández, Raquel; Prats, Elena; Jorrín-Novo, Jesús V.

    2010-01-01

    Plant pathogenic fungi cause important yield losses in crops. In order to develop efficient and environmental friendly crop protection strategies, molecular studies of the fungal biological cycle, virulence factors, and interaction with its host are necessary. For that reason, several approaches have been performed using both classical genetic, cell biology, and biochemistry and the modern, holistic, and high-throughput, omic techniques. This work briefly overviews the tools available for studying Plant Pathogenic Fungi and is amply focused on MS-based Proteomics analysis, based on original papers published up to December 2009. At a methodological level, different steps in a proteomic workflow experiment are discussed. Separate sections are devoted to fungal descriptive (intracellular, subcellular, extracellular) and differential expression proteomics and interactomics. From the work published we can conclude that Proteomics, in combination with other techniques, constitutes a powerful tool for providing important information about pathogenicity and virulence factors, thus opening up new possibilities for crop disease diagnosis and crop protection. PMID:20589070

  16. Certification of Total Arsenic in Blood and Urine Standard Reference Materials by Radiochemical Neutron Activation Analysis and Inductively Coupled Plasma - Mass Spectrometry

    PubMed Central

    Paul, Rick L.; Davis, W. Clay; Yu, Lee; Murphy, Karen E.; Guthrie, William F.; Leber, Dennis D.; Bryan, Colleen E.; Vetter, Thomas W.; Shakirova, Gulchekhra; Mitchell, Graylin; Kyle, David J.; Jarrett, Jeffery M.; Caldwell, Kathleen L.; Jones, Robert L.; Eckdahl, Steven; Wermers, Michelle; Maras, Melissa; Palmer, C. D.; Verostek, M.F.; Geraghty, C. M.; Steuerwald, Amy J.; Parsons, Patrick J.

    2015-01-01

    A newly developed procedure for determination of arsenic by radiochemical neutron activation analysis (RNAA) was used to measure arsenic at four levels in SRM 955c Toxic Elements in Caprine Blood and at two levels in SRM 2668 Toxic Elements in Frozen Human Urine for the purpose of providing mass concentration values for certification. Samples were freeze-dried prior to analysis followed by neutron irradiation for 3 h at a fluence rate of 1×1014cm−2s−1. After sample dissolution in perchloric and nitric acids, arsenic was separated from the matrix by extraction into zinc diethyldithiocarbamate in chloroform, and 76As quantified by gamma-ray spectroscopy. Differences in chemical yield and counting geometry between samples and standards were monitored by measuring the count rate of a 77As tracer added before sample dissolution. RNAA results were combined with inductively coupled plasma – mass spectrometry (ICP-MS) values from NIST and collaborating laboratories to provide certified values of (10.81 ± 0.54) μg/kg and (213.1 ± 0.73) μg/kg for SRM 2668 Levels I and II, and certified values of (21.66 ± 0.73) μg/kg, (52.7 ± 1.1) μg/kg, and (78.8 ± 4.9) μg/kg for SRM 955c Levels 2, 3, and 4 respectively. Because of discrepancies between values obtained by different methods for SRM 955c Level 1, an information value of < 5 μg/kg was assigned for this material. PMID:26300575

  17. Reference Materials

    NASA Astrophysics Data System (ADS)

    Merkus, Henk G.

    Reference materials for measurement of particle size and porosity may be used for calibration or qualification of instruments or for validation of operating procedures or operators. They cover a broad range of materials. On the one hand there are the certified reference materials, for which governmental institutes have certified one or more typical size or porosity values. Then, there is a large group of reference materials from commercial companies. And on the other hand there are typical products in a given line of industry, where size or porosity values come from the analysis laboratory itself or from some round-robin test in a group of industrial laboratories. Their regular application is essential for adequate quality control of particle size and porosity measurement, as required in e.g., ISO 17025 on quality management. In relation to this, some quality requirements for certification are presented.

  18. A “Proteomic Ruler” for Protein Copy Number and Concentration Estimation without Spike-in Standards*

    PubMed Central

    Wiśniewski, Jacek R.; Hein, Marco Y.; Cox, Jürgen; Mann, Matthias

    2014-01-01

    Absolute protein quantification using mass spectrometry (MS)-based proteomics delivers protein concentrations or copy numbers per cell. Existing methodologies typically require a combination of isotope-labeled spike-in references, cell counting, and protein concentration measurements. Here we present a novel method that delivers similar quantitative results directly from deep eukaryotic proteome datasets without any additional experimental steps. We show that the MS signal of histones can be used as a “proteomic ruler” because it is proportional to the amount of DNA in the sample, which in turn depends on the number of cells. As a result, our proteomic ruler approach adds an absolute scale to the MS readout and allows estimation of the copy numbers of individual proteins per cell. We compare our protein quantifications with values derived via the use of stable isotope labeling by amino acids in cell culture and protein epitope signature tags in a method that combines spike-in protein fragment standards with precise isotope label quantification. The proteomic ruler approach yields quantitative readouts that are in remarkably good agreement with results from the precision method. We attribute this surprising result to the fact that the proteomic ruler approach omits error-prone steps such as cell counting or protein concentration measurements. The proteomic ruler approach is readily applicable to any deep eukaryotic proteome dataset—even in retrospective analysis—and we demonstrate its usefulness with a series of mouse organ proteomes. PMID:25225357

  19. Proteomic profiling of the rat hypothalamus

    PubMed Central

    2012-01-01

    Background The hypothalamus plays a pivotal role in numerous mechanisms highly relevant to the maintenance of body homeostasis, such as the control of food intake and energy expenditure. Impairment of these mechanisms has been associated with the metabolic disturbances involved in the pathogenesis of obesity. Since rodent species constitute important models for metabolism studies and the rat hypothalamus is poorly characterized by proteomic strategies, we performed experiments aimed at constructing a two-dimensional gel electrophoresis (2-DE) profile of rat hypothalamus proteins. Results As a first step, we established the best conditions for tissue collection and protein extraction, quantification and separation. The extraction buffer composition selected for proteome characterization of rat hypothalamus was urea 7 M, thiourea 2 M, CHAPS 4%, Triton X-100 0.5%, followed by a precipitation step with chloroform/methanol. Two-dimensional (2-D) gels of hypothalamic extracts from four-month-old rats were analyzed; the protein spots were digested and identified by using tandem mass spectrometry and database query using the protein search engine MASCOT. Eighty-six hypothalamic proteins were identified, the majority of which were classified as participating in metabolic processes, consistent with the finding of a large number of proteins with catalytic activity. Genes encoding proteins identified in this study have been related to obesity development. Conclusion The present results indicate that the 2-DE technique will be useful for nutritional studies focusing on hypothalamic proteins. The data presented herein will serve as a reference database for studies testing the effects of dietary manipulations on hypothalamic proteome. We trust that these experiments will lead to important knowledge on protein targets of nutritional variables potentially able to affect the complex central nervous system control of energy homeostasis. PMID:22519962

  20. Imaging beyond the proteome

    PubMed Central

    Chang, Pamela V.; Bertozzi, Carolyn R.

    2013-01-01

    Imaging technologies developed in the early 20th century achieved contrast solely by relying on macroscopic and morphological differences between the tissues of interest and the surrounding tissues. Since then, there has been a movement toward imaging at the cellular and molecular level in order to visualize biological processes. This rapidly growing field is known as molecular imaging. In the last decade, many methodologies for imaging proteins have emerged. However, most of these approaches cannot be extended to imaging beyond the proteome. Here, we highlight some of the recently developed technologies that enable imaging of non-proteinaceous molecules in the cell: lipids, signalling molecules, inorganic ions, glycans, nucleic acids, small-molecule metabolites, and protein post-translational modifications such as phosphorylation and methylation. PMID:22801420

  1. Spectral library searching in proteomics.

    PubMed

    Griss, Johannes

    2016-03-01

    Spectral library searching has become a mature method to identify tandem mass spectra in proteomics data analysis. This review provides a comprehensive overview of available spectral library search engines and highlights their distinct features. Additionally, resources providing spectral libraries are summarized and tools presented that extend experimental spectral libraries by simulating spectra. Finally, spectrum clustering algorithms are discussed that utilize the same spectrum-to-spectrum matching algorithms as spectral library search engines and allow novel methods to analyse proteomics data.

  2. The Succinated Proteome

    SciTech Connect

    Merkley, Eric D.; Metz, Thomas O.; Smith, Richard D.; Baynes, John; Frizell, Norma

    2014-03-30

    Succination is a chemical modification of cysteine in protein by the Krebs cycle intermediate, fumarate, yielding S-(2-succino)cysteine (2SC). Intracellular fumarate concentration and succination of proteins are increased by hyperpolarization of the inner mitochondrial membrane, in concert with mitochondrial, endoplasmic reticulum (ER) and oxidative stress in adipocytes grown in high glucose medium and in adipose tissue in obesity and diabetes. Increased succination of proteins is also detected in the kidney of a fumarase conditional knock-out mouse which develops renal tumors. Keap1, the gatekeeper of the antioxidant response, was identified as a major succinated protein in renal cancer cells, suggesting that succination may play a role in activation of the antioxidant response. A wide range of proteins is subject to succination, including enzymes, adipokines, cytoskeletal proteins and ER chaperones with functional cysteine residues. There is also significant overlap between succinated and glutathionylated proteins, and with proteins containing cysteine residues that are readily oxidized to the sulfenic (cysteic) acid. Succination of adipocyte proteins is inhibited by uncouplers, which discharge the mitochondrial membrane potential (Δψm) and by ER stress inhibitors. 2SC serves as a biomarker of mitochondrial stress or dysfunction in chronic diseases, such as obesity, diabetes and cancer, and recent studies suggest that succination is a mechanistic link between mitochondrial dysfunction, oxidative and ER stress, and cellular progression toward apoptosis. In this article, we review the history of the succinated proteome and the challenges associated with measuring this non-enzymatic post-translational modification of proteins by proteomics approaches.

  3. Determination of halogens, with special reference to iodine, in geological and biological samples using pyrohydrolysis for preparation and inductively coupled plasma mass spectrometry and ion chromatography for measurement.

    PubMed

    Schnetger, B; Muramatsu, Y

    1996-11-01

    A method for determining iodine, bromine, chlorine and fluorine in geological and biological materials is described. In a quartz tube, solid material was heated to 1100 degrees C under a wet oxygen flow (pyrohydrolysis). By this process the halogens (I, Br, Cl, F) were separated from the matrix and then collected in a receiver solution. The chemical yield of iodine was determined by a radioactive tracer. ICP-MS and ion chromatographic measurements were used for the determination of the halogens. The method was optimized by investigating different experimental conditions. All four halogens can be trapped in the receiver solution from one combustion procedure. Precision and accuracy were evaluated by the analysis of environmental standard reference materials (rock, soil, milk, leaves, marine tissue). The concentrations in the materials analysed were in the ranges 0.006-50 mg kg-1 for I, 0.06-1300 mg kg-1 for Br, 50-1100 mg kg-1 for F and 400-11000 mg kg-1 for Cl. The lower values represent the practical detection limit of this method. The results obtained by the proposed method and the certified values are in good agreement.

  4. Determination of halogens, with special reference to iodine, in geological and biological samples using pyrohydrolysis for preparation and inductively coupled plasma mass spectrometry and ion chromatography for measurement.

    PubMed

    Schnetger, B; Muramatsu, Y

    1996-11-01

    A method for determining iodine, bromine, chlorine and fluorine in geological and biological materials is described. In a quartz tube, solid material was heated to 1100 degrees C under a wet oxygen flow (pyrohydrolysis). By this process the halogens (I, Br, Cl, F) were separated from the matrix and then collected in a receiver solution. The chemical yield of iodine was determined by a radioactive tracer. ICP-MS and ion chromatographic measurements were used for the determination of the halogens. The method was optimized by investigating different experimental conditions. All four halogens can be trapped in the receiver solution from one combustion procedure. Precision and accuracy were evaluated by the analysis of environmental standard reference materials (rock, soil, milk, leaves, marine tissue). The concentrations in the materials analysed were in the ranges 0.006-50 mg kg-1 for I, 0.06-1300 mg kg-1 for Br, 50-1100 mg kg-1 for F and 400-11000 mg kg-1 for Cl. The lower values represent the practical detection limit of this method. The results obtained by the proposed method and the certified values are in good agreement. PMID:8952450

  5. The proteome of human saliva

    NASA Astrophysics Data System (ADS)

    Griffin, Timothy J.

    2013-05-01

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

  6. Poroelastic references

    SciTech Connect

    Morency, Christina

    2014-12-12

    This file contains a list of relevant references on the Biot theory (forward and inverse approaches), the double-porosity and dual-permeability theory, and seismic wave propagation in fracture porous media, in RIS format, to approach seismic monitoring in a complex fractured porous medium such as Brady?s Geothermal Field.

  7. Ready Reference.

    ERIC Educational Resources Information Center

    Koltay, Emery

    2001-01-01

    Includes four articles that relate to ready reference, including a list of publishers' toll-free telephone numbers and Web sites; how to obtain an ISBN (International Standard Book Number) and an ISSN (International Standard Serial Number); and how to obtain an SAN (Standard Address Number), for organizations that are involved in the book…

  8. Enabling Proteomics Discovery Through Visual Analysis

    SciTech Connect

    Havre, Susan L.; Singhal, Mudita; Payne, Deborah A.; Lipton, Mary S.; Webb-Robertson, Bobbie-Jo M.

    2005-05-01

    With the completion of the Human Genome Project and the sequencing of large genomes, proteomics is the new big challenge. A proteome is the collection of all the proteins present in an organism at a given moment. Unlike the genome, the proteome is dynamic, changing continuously in response to tens of thousands of intra- and extra-cellular environmental signals. Proteomics is the study of proteomes under different conditions—for example, over time, under different environments, or in different disease states. Because proteins are the key actors in cellular processes and proteomics is the study of not one or two proteins at a time but whole proteomes, proteomics has a key role in revealing the complex processes of cells at a global or systems level. There are several high-throughput proteomics techniques; all generate data faster than the data can currently be analyzed. The tremendous size and complexity of the high-throughput experimental data make it very difficult to process and interpret. The success of proteomics will rely on high-throughput experimental techniques coupled with sophisticated visual analysis and data mining methods. This article presents the motivation for developing visual analysis tools for proteomic data and demonstrates their application to proteomics research with a visualization tool named Peptide Permutation and Protein Prediction, or PQuad. PQuad is a functioning visual analytic tool in operation at the Pacific Northwest National Laboratory for the study of systems biology. PQuad supports the exploration of proteins identified by proteomic techniques in the context of supplemental biological information.

  9. Mass Spectrometry-based Proteomics in Acute Respiratory Distress Syndrome: A Powerful Modality for Pulmonary Precision Medicine

    PubMed Central

    Xu, Xue-Feng; Dai, Hua-Ping; Li, Yan-Ming; Xiao, Fei; Wang, Chen

    2016-01-01

    Objective: Acute respiratory distress syndrome (ARDS) is an acute and lethal clinical syndrome that is characterized by hypoxemic respiratory failure and diffuse alveolar inflammatory damage. This review aimed to search and discuss the mass spectrometry (MS)-based proteomic studies on different subsets of ARDS patients. Data Sources: Original research articles were collected from the PubMed database published in English up to December 2015. Study Selection: The literature search was done using the term “(acute lung injury OR acute respiratory distress syndrome) AND (proteomics OR proteome OR mass spectrum OR differential in-gel electrophoresis OR two-dimensional polyacrylamide gel electrophoresis)”. Related original research articles were included and were carefully analyzed. Results: Eight original proteomic researches on ARDS patients were found. The common proteomic modalities were two-dimensional (2D) high-performance liquid chromatography-based electronic spray ion-MS/MS and 2D-polyacrylamide gel electrophoresis/differential in-gel electrophoresis-based matrix-assisted laser desorption ionization-time of flight/MS. They compared the proteome between ARDS patients and normal controls and analyzed the dynamic changes of proteome at different ARDS stages or severity. The disturbed proteome in ARDS patients includes plasma acute-phase proteins, inflammatory/immune-associated proteins, and coagulation proteins. Conclusions: Although several previous studies have provided some useful information about the lung proteome in ARDS patients and gained several interesting disease-associated biomarkers, clinical proteomic studies in ARDS patients are still in the initial stage. An increased cooperation is still needed to establish a global and faithful database containing disease-specific proteome from the largest ARDS subsets. PMID:27647196

  10. Pharmacokinetics of tildipirosin in porcine plasma, lung tissue, and bronchial fluid and effects of test conditions on in vitro activity against reference strains and field isolates of Actinobacillus pleuropneumoniae.

    PubMed

    Rose, M; Menge, M; Bohland, C; Zschiesche, E; Wilhelm, C; Kilp, S; Metz, W; Allan, M; Röpke, R; Nürnberger, M

    2013-04-01

    The pharmacokinetics of tildipirosin (Zuprevo(®) 40 mg/mL solution for injection for pigs), a novel 16-membered-ring macrolide for the treatment for swine respiratory disease (SRD), was investigated in studies collecting blood plasma and postmortem samples of lung tissue and bronchial fluid (BF) from swine. In view of factors influencing the in vitro activity of macrolides, and for the interpretation of tildipirosin pharmacokinetics in relation to minimum inhibitory concentrations (MIC), additional experiments were conducted to study the effects of pH, carbon dioxide-enriched atmosphere, buffers, and serum on tildipirosin MICs for various reference strains and Actinobacillus (A.) pleuropneumoniae field isolates. After single intramuscular (i.m.) injection at 4 mg/kg body weight, maximum plasma concentration (Cmax) was 0.9 μg/mL observed within 23 min (Tmax ). Mean residence time from the time of dosing to the time of last measurable concentration (MRTlast) and terminal half-life (T1/2) both were about 4 days. A dose-response relationship with no significant sex effect is observed for area under the plasma concentration-time curve from time 0 to the last sampling time with a quantifiable drug concentration (AUClast) over the range of doses up to 6 mg/kg. However, linear dose proportionality could not be proven with statistical methods. The time-concentration profile of tildipirosin in BF and lung far exceeded that in blood plasma. In lung, tildipirosin concentrations reached 3.1 μg/g at 2 h, peaked at 4.3 μg/g at day 1, and slowly declined to 0.8 μg/g at day 17. In BF, tildipirosin levels were 14.3, 7.0, and 6.5 μg/g at days 5, 10, and 14. T1/2 in lung was ∼7 days. Tildipirosin is rapidly and extensively distributed to the respiratory tract followed by slow elimination. Culture media pH and carbon dioxide-enriched atmosphere (CO2 -EA) had a marked impact on in vitro activity of tildipirosin in reference strains of various rapidly growing aerobic and

  11. On the privacy risks of sharing clinical proteomics data.

    PubMed

    Li, Sujun; Bandeira, Nuno; Wang, Xiaofeng; Tang, Haixu

    2016-01-01

    Although the privacy issues in human genomic studies are well known, the privacy risks in clinical proteomic data have not been thoroughly studied. As a proof of concept, we reported a comprehensive analysis of the privacy risks in clinical proteomic data. It showed that a small number of peptides carrying the minor alleles (referred to as the minor allelic peptides) at non-synonymous single nucleotide polymorphism (nsSNP) sites can be identified in typical clinical proteomic datasets acquired from the blood/serum samples of individual patient, from which the patient can be identified with high confidence. Our results suggested the presence of significant privacy risks in raw clinical proteomic data. However, these risks can be mitigated by a straightforward pre-processing step of the raw data that removing a very small fraction (0.1%, 7.14 out of 7,504 spectra on average) of MS/MS spectra identified as the minor allelic peptides, which has little or no impact on the subsequent analysis (and re-use) of these datasets. PMID:27595046

  12. On the privacy risks of sharing clinical proteomics data

    PubMed Central

    Li, Sujun; Bandeira, Nuno; Wang, Xiaofeng; Tang, Haixu

    2016-01-01

    Although the privacy issues in human genomic studies are well known, the privacy risks in clinical proteomic data have not been thoroughly studied. As a proof of concept, we reported a comprehensive analysis of the privacy risks in clinical proteomic data. It showed that a small number of peptides carrying the minor alleles (referred to as the minor allelic peptides) at non-synonymous single nucleotide polymorphism (nsSNP) sites can be identified in typical clinical proteomic datasets acquired from the blood/serum samples of individual patient, from which the patient can be identified with high confidence. Our results suggested the presence of significant privacy risks in raw clinical proteomic data. However, these risks can be mitigated by a straightforward pre-processing step of the raw data that removing a very small fraction (0.1%, 7.14 out of 7,504 spectra on average) of MS/MS spectra identified as the minor allelic peptides, which has little or no impact on the subsequent analysis (and re-use) of these datasets. PMID:27595046

  13. On the privacy risks of sharing clinical proteomics data

    PubMed Central

    Li, Sujun; Bandeira, Nuno; Wang, Xiaofeng; Tang, Haixu

    2016-01-01

    Although the privacy issues in human genomic studies are well known, the privacy risks in clinical proteomic data have not been thoroughly studied. As a proof of concept, we reported a comprehensive analysis of the privacy risks in clinical proteomic data. It showed that a small number of peptides carrying the minor alleles (referred to as the minor allelic peptides) at non-synonymous single nucleotide polymorphism (nsSNP) sites can be identified in typical clinical proteomic datasets acquired from the blood/serum samples of individual patient, from which the patient can be identified with high confidence. Our results suggested the presence of significant privacy risks in raw clinical proteomic data. However, these risks can be mitigated by a straightforward pre-processing step of the raw data that removing a very small fraction (0.1%, 7.14 out of 7,504 spectra on average) of MS/MS spectra identified as the minor allelic peptides, which has little or no impact on the subsequent analysis (and re-use) of these datasets.

  14. Hepatic SILAC proteomic data from PANDER transgenic model.

    PubMed

    Athanason, Mark G; Stevens, Stanley M; Burkhardt, Brant R

    2016-12-01

    This article contains raw and processed data related to research published in "Quantitative Proteomic Profiling Reveals Hepatic Lipogenesis and Liver X Receptor Activation in the PANDER Transgenic Model" (M.G. Athanason, W.A. Ratliff, D. Chaput, C.B. MarElia, M.N. Kuehl, S.M., Jr. Stevens, B.R. Burkhardt (2016)) [1], and was generated by "spike-in" SILAC-based proteomic analysis of livers obtained from the PANcreatic-Derived factor (PANDER) transgenic mouse (PANTG) under various metabolic conditions [1]. The mass spectrometry output of the PANTG and wild-type B6SJLF mice liver tissue and resulting proteome search from MaxQuant 1.2.2.5 employing the Andromeda search algorithm against the UniprotKB reference database for Mus musculus has been deposited to the ProteomeXchange Consortium (http://www.proteomexchange.org) via the PRIDE partner repository with dataset identifiers PRIDE: PXD004171 and doi:10.6019/PXD004171. Protein ratio values representing PANTG/wild-type obtained by MaxQuant analysis were input into the Perseus processing suite to determine statistical significance using the Significance A outlier test (p<0.05). Differentially expressed proteins using this approach were input into Ingenuity Pathway Analysis to determined altered pathways and upstream regulators that were altered in PANTG mice.

  15. Proteomics and Phosphoproteomics Analysis of Human Lens Fiber Cell Membranes

    PubMed Central

    Wang, Zhen; Han, Jun; David, Larry L.; Schey, Kevin L.

    2013-01-01

    Purpose. The human lens fiber cell insoluble membrane fraction contains important membrane proteins, cytoskeletal proteins, and cytosolic proteins that are strongly associated with the membrane. The purpose of this study was to characterize the lens fiber cell membrane proteome and phosphoproteome from human lenses. Methods. HPLC-mass spectrometry–based multidimensional protein identification technology (MudPIT), without or with phosphopeptide enrichment, was applied to study the proteome and phosphoproteome of lens fiber cell membranes, respectively. Results. In total, 951 proteins were identified, including 379 integral membrane and membrane-associated proteins. Enriched gene categories and pathways based on the proteomic analysis include carbohydrate metabolism (glycolysis/gluconeogenesis, pentose phosphate pathway, pyruvate metabolism), proteasome, cell-cell signaling and communication (GTP binding, gap junction, focal adhesion), glutathione metabolism, and actin regulation. The combination of TiO2 phosphopeptide enrichment and MudPIT analysis revealed 855 phosphorylation sites on 271 proteins, including 455 phosphorylation sites that have not been previously identified. PKA, PKC, CKII, p38MAPK, and RSK are predicted as the major kinases for phosphorylation on the sites identified in the human lens membrane fraction. Conclusions. The results presented herein significantly expand the characterized proteome and phosphoproteome of the human lens fiber cell and provide a valuable reference for future research in studies of lens development and disease. PMID:23349431

  16. Hepatic SILAC proteomic data from PANDER transgenic model.

    PubMed

    Athanason, Mark G; Stevens, Stanley M; Burkhardt, Brant R

    2016-12-01

    This article contains raw and processed data related to research published in "Quantitative Proteomic Profiling Reveals Hepatic Lipogenesis and Liver X Receptor Activation in the PANDER Transgenic Model" (M.G. Athanason, W.A. Ratliff, D. Chaput, C.B. MarElia, M.N. Kuehl, S.M., Jr. Stevens, B.R. Burkhardt (2016)) [1], and was generated by "spike-in" SILAC-based proteomic analysis of livers obtained from the PANcreatic-Derived factor (PANDER) transgenic mouse (PANTG) under various metabolic conditions [1]. The mass spectrometry output of the PANTG and wild-type B6SJLF mice liver tissue and resulting proteome search from MaxQuant 1.2.2.5 employing the Andromeda search algorithm against the UniprotKB reference database for Mus musculus has been deposited to the ProteomeXchange Consortium (http://www.proteomexchange.org) via the PRIDE partner repository with dataset identifiers PRIDE: PXD004171 and doi:10.6019/PXD004171. Protein ratio values representing PANTG/wild-type obtained by MaxQuant analysis were input into the Perseus processing suite to determine statistical significance using the Significance A outlier test (p<0.05). Differentially expressed proteins using this approach were input into Ingenuity Pathway Analysis to determined altered pathways and upstream regulators that were altered in PANTG mice. PMID:27642623

  17. Reproducibility of Differential Proteomic Technologies in CPTAC Fractionated Xenografts

    PubMed Central

    2015-01-01

    The NCI Clinical Proteomic Tumor Analysis Consortium (CPTAC) employed a pair of reference xenograft proteomes for initial platform validation and ongoing quality control of its data collection for The Cancer Genome Atlas (TCGA) tumors. These two xenografts, representing basal and luminal-B human breast cancer, were fractionated and analyzed on six mass spectrometers in a total of 46 replicates divided between iTRAQ and label-free technologies, spanning a total of 1095 LC–MS/MS experiments. These data represent a unique opportunity to evaluate the stability of proteomic differentiation by mass spectrometry over many months of time for individual instruments or across instruments running dissimilar workflows. We evaluated iTRAQ reporter ions, label-free spectral counts, and label-free extracted ion chromatograms as strategies for data interpretation (source code is available from http://homepages.uc.edu/~wang2x7/Research.htm). From these assessments, we found that differential genes from a single replicate were confirmed by other replicates on the same instrument from 61 to 93% of the time. When comparing across different instruments and quantitative technologies, using multiple replicates, differential genes were reproduced by other data sets from 67 to 99% of the time. Projecting gene differences to biological pathways and networks increased the degree of similarity. These overlaps send an encouraging message about the maturity of technologies for proteomic differentiation. PMID:26653538

  18. Method development for proteome stabilization in human saliva.

    PubMed

    Xiao, Hua; Wong, David T W

    2012-04-13

    Human saliva is a biological fluid with emerging early detection and diagnostic potentials. However, the salivary proteome suffers from rapid degradation and thus compromises its translational and clinical utilities. Therefore, easy, reliable and practical methods are urgently required for the storage of human saliva samples. In this study, saliva samples from healthy subjects were collected and stored at room temperature (RT) and 4 °C for different lengths of time with and without specific protein stabilization treatments. SDS-PAGE was run to compare the protein profiling between samples. Reference proteins, β-actin and interleukin-1 β (IL1β), were chosen to evaluate salivary protein stability. Immunoassay was used for the detection of these target proteins. All data was compared with the positive control that had been kept at -80 °C. The results show that the salivary proteome that has been stored at 4 °C with added protease inhibitors was stable for approximately two weeks without significant degradation. By adding ethanol to the samples, the salivary proteome was stabilized at RT. After optimization, a simple, robust and convenient method is developed for the stabilization of proteins in human saliva that does not affect the downstream translational and clinical applications. The salivary proteome could be stabilized without significant degradation by adding ethanol at RT for about two weeks. This optimized method could greatly accelerate the clinical usage of saliva for future diagnosis.

  19. Proteomic analysis and discovery using affinity proteomics and mass spectrometry.

    PubMed

    Olsson, Niclas; Wingren, Christer; Mattsson, Mikael; James, Peter; O'Connell, David; Nilsson, Fredrik; Cahill, Dolores J; Borrebaeck, Carl A K

    2011-10-01

    Antibody-based microarrays are a rapidly evolving affinity-proteomic methodology that recently has shown great promise in clinical applications. The resolution of these proteomic analyses is, however, directly related to the number of data-points, i.e. antibodies, included on the array. Currently, this is a key bottleneck because of limited availability of numerous highly characterized antibodies. Here, we present a conceptually new method, denoted global proteome survey, opening up the possibility to probe any proteome in a species-independent manner while still using a limited set of antibodies. We use context-independent-motif-specific antibodies directed against short amino acid motifs, where each motif is present in up to a few hundred different proteins. First, the digested proteome is exposed to these antibodies, whereby motif-containing peptides are enriched, which then are detected and identified by mass spectrometry. In this study, we profiled extracts from human colon tissue, yeast cells lysate, and mouse liver tissue to demonstrate proof-of-concept.

  20. Proteomic analysis of seminal fluid from men exhibiting oxidative stress

    PubMed Central

    2013-01-01

    Background Seminal plasma serves as a natural reservoir of antioxidants. It helps to remove excessive formation of reactive oxygen species (ROS) and consequently, reduce oxidative stress. Proteomic profiling of seminal plasma proteins is important to understand the molecular mechanisms underlying oxidative stress and sperm dysfunction in infertile men. Methods This prospective study consisted of 52 subjects: 32 infertile men and 20 healthy donors. Once semen and oxidative stress parameters were assessed (ROS, antioxidant concentration and DNA damage), the subjects were categorized into ROS positive (ROS+) or ROS negative (ROS-). Seminal plasma from each group was pooled and subjected to proteomics analysis. In-solution digestion and protein identification with liquid chromatography tandem mass spectrometry (LC-MS/MS), followed by bioinformatics analyses was used to identify and characterize potential biomarker proteins. Results A total of 14 proteins were identified in this analysis with 7 of these common and unique proteins were identified in both the ROS+ and ROS- groups through MASCOT and SEQUEST analyses, respectively. Prolactin-induced protein was found to be more abundantly present in men with increased levels of ROS. Gene ontology annotations showed extracellular distribution of proteins with a major role in antioxidative activity and regulatory processes. Conclusions We have identified proteins that help protect against oxidative stress and are uniquely present in the seminal plasma of the ROS- men. Men exhibiting high levels of ROS in their seminal ejaculate are likely to exhibit proteins that are either downregulated or oxidatively modified, and these could potentially contribute to male infertility. PMID:24004880

  1. Proteomics in the fruit tree science arena: new insights into fruit defense, development, and ripening.

    PubMed

    Molassiotis, Athanassios; Tanou, Georgia; Filippou, Panagiota; Fotopoulos, Vasileios

    2013-06-01

    Fruit tree crops are agricultural commodities of high economic importance, while fruits also represent one of the most vital components of the human diet. Therefore, a great effort has been made to understand the molecular mechanisms covering fundamental biological processes in fruit tree physiology and fruit biology. Thanks to the development of cutting-edge "omics" technologies such as proteomic analysis, scientists now have powerful tools to support traditional fruit tree research. Such proteomic analyses are establishing high-density 2DE reference maps and peptide mass fingerprint databases that can lead fruit science into a new postgenomic research era. Here, an overview of the application of proteomics in key aspects of fruit tree physiology as well as in fruit biology, including defense responses to abiotic and biotic stress factors, is presented. A panoramic view of ripening-related proteins is also discussed, as an example of proteomic application in fruit science.

  2. Comparative proteomics of leaf, stem, and root tissues of synthetic Brassica napus.

    PubMed

    Albertin, Warren; Langella, Olivier; Joets, Johann; Négroni, Luc; Zivy, Michel; Damerval, Catherine; Thiellement, Hervé

    2009-02-01

    Comparative proteomics was applied to three vegetative organs of Brassica napus, the leaf, stem, and root using 2-DE. Among the >1600 analyzed spots, 43% were found to be common to all three organs, suggesting the existence of a "basal" or ubiquitous proteome composed of housekeeping proteins. The green organs, leaf, and stem, were closely related (approximately 80% common spots) while the root displayed more organ-specific polypeptides (approximately 10%). Reference maps were established using MS, allowing the identification of 93, 385, and 266 proteins in leaf, stem, and root proteomes, respectively. Bioinformatic analyses were also performed; in silico functional categorization and cellular localization allow obtaining a precise picture of the cell molecular network within vegetative organs. These proteome maps can be explored using the PROTICdb software at the following address: http://bioinformatique.moulon.inra.fr/proticdb/web_view/.

  3. Aptamer-Based Multiplexed Proteomic Technology for Biomarker Discovery

    PubMed Central

    Gold, Larry; Ayers, Deborah; Bertino, Jennifer; Bock, Christopher; Bock, Ashley; Brody, Edward N.; Carter, Jeff; Dalby, Andrew B.; Eaton, Bruce E.; Fitzwater, Tim; Flather, Dylan; Forbes, Ashley; Foreman, Trudi; Fowler, Cate; Gawande, Bharat; Goss, Meredith; Gunn, Magda; Gupta, Shashi; Halladay, Dennis; Heil, Jim; Heilig, Joe; Hicke, Brian; Husar, Gregory; Janjic, Nebojsa; Jarvis, Thale; Jennings, Susan; Katilius, Evaldas; Keeney, Tracy R.; Kim, Nancy; Koch, Tad H.; Kraemer, Stephan; Kroiss, Luke; Le, Ngan; Levine, Daniel; Lindsey, Wes; Lollo, Bridget; Mayfield, Wes; Mehan, Mike; Mehler, Robert; Nelson, Sally K.; Nelson, Michele; Nieuwlandt, Dan; Nikrad, Malti; Ochsner, Urs; Ostroff, Rachel M.; Otis, Matt; Parker, Thomas; Pietrasiewicz, Steve; Resnicow, Daniel I.; Rohloff, John; Sanders, Glenn; Sattin, Sarah; Schneider, Daniel; Singer, Britta; Stanton, Martin; Sterkel, Alana; Stewart, Alex; Stratford, Suzanne; Vaught, Jonathan D.; Vrkljan, Mike; Walker, Jeffrey J.; Watrobka, Mike; Waugh, Sheela; Weiss, Allison; Wilcox, Sheri K.; Wolfson, Alexey; Wolk, Steven K.; Zhang, Chi; Zichi, Dom

    2010-01-01

    Background The interrogation of proteomes (“proteomics”) in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine. Methodology/Principal Findings We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 µL of serum or plasma). Our current assay measures 813 proteins with low limits of detection (1 pM median), 7 logs of overall dynamic range (∼100 fM–1 µM), and 5% median coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding signature of DNA aptamer concentrations, which is quantified on a DNA microarray. Our assay takes advantage of the dual nature of aptamers as both folded protein-binding entities with defined shapes and unique nucleotide sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to rapidly discover unique protein signatures characteristic of various disease states. Conclusions/Significance We describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of

  4. Proteomic approaches to bacterial differentiation

    SciTech Connect

    Norbeck, Angela D.; Callister, Stephen J.; Monroe, Matthew E.; Jaitly, Navdeep; Elias, Dwayne A.; Lipton, Mary S.; Smith, Richard D.

    2006-12-01

    While genomic approaches have been applied for the detection and identification of individual bacteria within microbial communities, analogous proteomics approaches have been effectively precluded due to their inherent complexity. An in silico assessment of peptides that could potentially be present in the proteomes of artificial simple and complex communities was performed to evaluate the effect of proteome complexity on species detection. A mass spectrometry-based proteomics approach was employed to experimentally detect and validate the predicted tryptic peptides initially identified as distinctive within the simple community. An assessment of peptide distinctiveness and the potential for mapping to a particular bacterium within a community was made throughout each step of the study. A second in silico assessment of peptide distinctiveness for a complex community of 25 microorganisms was conducted to investigate the levels of instrumental performance that would be required to experimentally detect these peptides, as well as how performance varied with complexity (e.g., the number of different microorganisms). The experimental data for a simple community showed that it is feasible to predict, observe, and to quantify distinctive peptides from one organism in the presence of at least a 100-fold greater abundance of another, thus yielding putative markers for identifying a bacterium of interest. This work represents a first step towards quantitative proteomic characterization of complex microbial communities and the possible development of community wide markers of perturbations to such communities.

  5. The proteome browser web portal.

    PubMed

    Goode, Robert J A; Yu, Simon; Kannan, Anitha; Christiansen, Jeffrey H; Beitz, Anthony; Hancock, William S; Nice, Edouard; Smith, A Ian

    2013-01-01

    In 2010, the Human Proteome Organization launched the Human Proteome Project (HPP), aimed at identifying and characterizing the proteome of the human body. To support complete coverage, one arm of the project will take a gene- or chromosomal-centric strategy (C-HPP) aimed at identifying at least one protein product from each protein-coding gene. Despite multiple large international biological databases housing genomic and protein data, there is currently no single system that integrates updated pertinent information from each of these data repositories and assembles the information into a searchable format suitable for the type of global proteomics effort proposed by the C-HPP. We have undertaken the goal of producing a data integration and analysis software system and browser for the C-HPP effort and of making data collections from this resource discoverable through metadata repositories, such as Australian National Data Service's Research Data Australia. Here we present our vision and progress toward the goal of developing a comprehensive data integration and analysis software tool that provides a snapshot of currently available proteomic related knowledge around each gene product, which will ultimately assist in analyzing biological function and the study of human physiology in health and disease.

  6. Proteomic insights into floral biology.

    PubMed

    Li, Xiaobai; Jackson, Aaron; Xie, Ming; Wu, Dianxing; Tsai, Wen-Chieh; Zhang, Sheng

    2016-08-01

    The flower is the most important biological structure for ensuring angiosperms reproductive success. Not only does the flower contain critical reproductive organs, but the wide variation in morphology, color, and scent has evolved to entice specialized pollinators, and arguably mankind in many cases, to ensure the successful propagation of its species. Recent proteomic approaches have identified protein candidates related to these flower traits, which has shed light on a number of previously unknown mechanisms underlying these traits. This review article provides a comprehensive overview of the latest advances in proteomic research in floral biology according to the order of flower structure, from corolla to male and female reproductive organs. It summarizes mainstream proteomic methods for plant research and recent improvements on two dimensional gel electrophoresis and gel-free workflows for both peptide level and protein level analysis. The recent advances in sequencing technologies provide a new paradigm for the ever-increasing genome and transcriptome information on many organisms. It is now possible to integrate genomic and transcriptomic data with proteomic results for large-scale protein characterization, so that a global understanding of the complex molecular networks in flower biology can be readily achieved. 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. PMID:26945514

  7. Mammalian plasma membrane proteins as potential biomarkers and drug targets.

    PubMed

    Rucevic, Marijana; Hixson, Douglas; Josic, Djuro

    2011-06-01

    Defining the plasma membrane proteome is crucial to understand the role of plasma membrane in fundamental biological processes. Change in membrane proteins is one of the first events that take place under pathological conditions, making plasma membrane proteins a likely source of potential disease biomarkers with prognostic or diagnostic potential. Membrane proteins are also potential targets for monoclonal antibodies and other drugs that block receptors or inhibit enzymes essential to the disease progress. Despite several advanced methods recently developed for the analysis of hydrophobic proteins and proteins with posttranslational modifications, integral membrane proteins are still under-represented in plasma membrane proteome. Recent advances in proteomic investigation of plasma membrane proteins, defining their roles as diagnostic and prognostic disease biomarkers and as target molecules in disease treatment, are presented.

  8. Proteome of Hydra Nematocyst*

    PubMed Central

    Balasubramanian, Prakash G.; Beckmann, Anna; Warnken, Uwe; Schnölzer, Martina; Schüler, Andreas; Bornberg-Bauer, Erich; Holstein, Thomas W.; Özbek, Suat

    2012-01-01

    Stinging cells or nematocytes of jellyfish and other cnidarians represent one of the most poisonous and sophisticated cellular inventions in animal evolution. This ancient cell type is unique in containing a giant secretory vesicle derived from the Golgi apparatus. The organelle structure within the vesicle comprises an elastically stretched capsule (nematocyst) to which a long tubule is attached. During exocytosis, the barbed part of the tubule is accelerated with >5 million g in <700 ns, enabling a harpoon-like discharge (Nüchter, T., Benoit, M., Engel, U., Ozbek, S., and Holstein, T. W. (2006) Curr. Biol. 16, R316–R318). Hitherto, the molecular components responsible for the organelle's biomechanical properties were largely unknown. Here, we describe the proteome of nematocysts from the freshwater polyp Hydra magnipapillata. Our analysis revealed an unexpectedly complex secretome of 410 proteins with venomous and lytic but also adhesive or fibrous properties. In particular, the insoluble fraction of the nematocyst represents a functional extracellular matrix structure of collagenous and elastic nature. This finding suggests an evolutionary scenario in which exocytic vesicles harboring a venomous secretome assembled a sophisticated predatory structure from extracellular matrix motif proteins. PMID:22291027

  9. High-throughput proteomics

    NASA Astrophysics Data System (ADS)

    Lesley, Scott A.; Nasoff, Marc; Kreusch, Andreas; Spraggon, Glen

    2001-04-01

    Proteomics has become a major focus as researchers attempt to understand the vast amount of genomic information. Protein complexity makes identifying and understanding gene function inherently difficult. The challenge of studying proteins in a global way is driving the development of new technologies for systematic and comprehensive analysis of protein structure and function. We are addressing this challenge through instrumentation and approaches to rapidly express, purify, crystallize, and mutate large numbers of human gene products. Our approach applies the principles of HTS technologies commonly used in pharmaceutical development. Genes are cloned, expressed, and purified in parallel to achieve a throughput potential of hundreds per day. Our instrumentation allows us to produce tens of milligrams of protein from 96 separate clones simultaneously. Purified protein is used for several applications including a high-throughput crystallographic screening approach for structure determination using automated image analysis. To further understand protein function, we are integrating a mutagenesis and screening approach. By combining these key technologies, we hope to provide a fundamental basis for understanding gene function at the protein level.

  10. Use of proteomic methods in the analysis of human body fluids in Alzheimer research.

    PubMed

    Zürbig, Petra; Jahn, Holger

    2012-12-01

    Proteomics is the study of the entire population of proteins and peptides in an organism or a part of it, such as a cell, tissue, or fluids like cerebrospinal fluid, plasma, serum, urine, or saliva. It is widely assumed that changes in the composition of the proteome may reflect disease states and provide clues to its origin, eventually leading to targets for new treatments. The ability to perform large-scale proteomic studies now is based jointly on recent advances in our analytical methods. Separation techniques like CE and 2DE have developed and matured. Detection methods like MS have also improved greatly in the last 5 years. These developments have also driven the fields of bioinformatics, needed to deal with the increased data production and systems biology. All these developing methods offer specific advantages but also come with certain limitations. This review describes the different proteomic methods used in the field, their limitations, and their possible pitfalls. Based on a literature search in PubMed, we identified 112 studies that applied proteomic techniques to identify biomarkers for Alzheimer disease. This review describes the results of these studies on proteome changes in human body fluids of Alzheimer patients reviewing the most important studies. We extracted a list of 366 proteins and peptides that were identified by these studies as potential targets in Alzheimer research.

  11. Heart-brain signaling in patent foramen ovale-related stroke: differential plasma proteomic expression patterns revealed with a 2-pass liquid chromatography-tandem mass spectrometry discovery workflow.

    PubMed

    Lopez, Mary F; Sarracino, David A; Vogelsang, Maryann; Sutton, Jennifer N; Athanas, Michael; Krastins, Bryan; Garces, Alejandra; Prakash, Amol; Peterman, Scott; Demirjian, Zareh; Inglessis-Azuaje, Ignacio; Feeney, Kathleen; Elia, Mikaela; McMullin, David; Dec, G William; Palacios, Igor; Lo, Eng H; Buonanno, Ferdinand; Ning, MingMing

    2012-12-01

    Patent foramen ovale (PFO) is highly prevalent and associated with more than 150,000 strokes per year. Traditionally, it is thought that PFOs facilitate strokes by allowing venous clots to travel directly to the brain. However, only a small portion of PFO stroke patients have a known tendency to form blood clots, and the optimal treatment for this multiorgan disease is unclear. Therefore, mapping the changes in systemic circulation of PFO-related stroke is crucial in understanding the pathophysiology to individualize the best clinical treatment for each patient. We initiated a study using a novel quantitative, 2-pass discovery workflow using high-resolution liquid chromatography-mass spectrometry/mass spectrometry coupled with label-free analysis to track protein expression in PFO patients before and after endovascular closure of the PFO. Using this approach, we were able to demonstrate quantitative differences in protein expression between both PFO-related and non-PFO-related ischemic stroke groups as well as before and after PFO closure. As an initial step in understanding the molecular landscape of PFO-related physiology, our methods have yielded biologically relevant information on the synergistic and functional redundancy of various cell-signaling molecules with respect to PFO circulatory physiology. The resulting protein expression patterns were related to canonical pathways including prothrombin activation, atherosclerosis signaling, acute-phase response, LXR/RXR activation, and coagulation system. In particular, after PFO closure, numerous proteins demonstrated reduced expression in stroke-related canonical pathways such as acute inflammatory response and coagulation signaling. These findings demonstrate the feasibility and robustness of using a proteomic approach for biomarker discovery to help gauge therapeutic efficacy in stroke.

  12. Proteomics technology in systems biology.

    PubMed

    Smith, Jeffrey C; Figeys, Daniel

    2006-08-01

    It has now become apparent that a full understanding of a biological process (e.g. a disease state) is only possible if all biomolecular interactions are taken into account. Systems biology works towards understanding the intricacies of cellular life through the collaborative efforts of biologists, chemists, mathematicians and computer scientists and recently, a number of laboratories around the world have embarked upon such research agendas. The fields of genomics and proteomics are foundational in systems biology studies and a great deal of research is currently being conducted in each worldwide. Moreover, many technological advances (particularly in mass spectrometry) have led to a dramatic rise in the number of proteomic studies over the past two decades. This short review summarizes a selection of technological innovations in proteomics that contribute to systems biology studies. PMID:16880956

  13. Plasma and liver proteomic analysis of 3Z-3-[(1H-pyrrol-2-yl)-methylidene]-1-(1-piperidinylmethyl)-1,3-2H-indol-2-one-induced hepatotoxicity in Wistar rats.

    PubMed

    Wang, Ying; Yang, Baohua; Wu, Chunqi; Zheng, Zhibing; Yuan, Ye; Hu, ZhongHui; Ma, HuaZhi; Li, Song; Liao, Mingyang; Wang, Quanjun

    2010-08-01

    3Z-3-[(1H-pyrrol-2-yl)-methylidene]-1-(1-piperidinylmethyl)-1,3-2H-indol-2-one (Z24), a synthetic anti-angiogenic compound, inhibits the growth and metastasis of certain tumors. Previous works have shown that Z24 induces hepatotoxicity in rodents. We examined the hepatotoxic mechanism of Z24 at the protein level and looked for potential biomarkers. We used 2-DE and MALDI-TOF/TOF MS to analyze alternatively expressed proteins in rat liver and plasma after Z24 administration. We also examined apoptosis in rat liver and measured levels of intramitochondrial ROS and NAD(P)H redox in liver cells. We found that 22 nonredundant proteins in the liver and 11 in the plasma were differentially expressed. These proteins were involved in several important metabolic pathways, including carbohydrate, lipid, amino acid, and energy metabolism, biotransformation, apoptosis, etc. Apoptosis in rat liver was confirmed with the terminal deoxynucleotidyl transferase dUTP-nick end labeling assay. In mitochondria, Z24 increased the ROS and decreased the NAD(P)H levels. Thus, inhibition of carbohydrate aerobic oxidation, fatty acid beta-oxidation, and oxidative phosphorylation is a potential mechanism of Z24-induced hepatotoxicity, resulting in mitochondrial dysfunction and apoptosis-mediated cell death. In addition, fetub protein and argininosuccinate synthase in plasma may be potential biomarkers of Z24-induced hepatotoxicity.

  14. Determination of burn patient outcome by large-scale quantitative discovery proteomics

    PubMed Central

    Finnerty, Celeste C.; Jeschke, Marc G.; Qian, Wei-Jun; Kaushal, Amit; Xiao, Wenzhong; Liu, Tao; Gritsenko, Marina A.; Moore, Ronald J.; Camp, David G.; Moldawer, Lyle L.; Elson, Constance; Schoenfeld, David; Gamelli, Richard; Gibran, Nicole; Klein, Matthew; Arnoldo, Brett; Remick, Daniel; Smith, Richard D.; Davis, Ronald; Tompkins, Ronald G.; Herndon, David N.

    2013-01-01

    Objective Emerging proteomics techniques can be used to establish proteomic outcome signatures and to identify candidate biomarkers for survival following traumatic injury. We applied high-resolution liquid chromatography-mass spectrometry (LC-MS) and multiplex cytokine analysis to profile the plasma proteome of survivors and non-survivors of massive burn injury to determine the proteomic survival signature following a major burn injury. Design Proteomic discovery study. Setting Five burn hospitals across the U.S. Patients Thirty-two burn patients (16 non-survivors and 16 survivors), 19–89 years of age, were admitted within 96 h of injury to the participating hospitals with burns covering >20% of the total body surface area and required at least one surgical intervention. Interventions None. Measurements and Main Results We found differences in circulating levels of 43 proteins involved in the acute phase response, hepatic signaling, the complement cascade, inflammation, and insulin resistance. Thirty-two of the proteins identified were not previously known to play a role in the response to burn. IL-4, IL-8, GM-CSF, MCP-1, and β2-microglobulin correlated well with survival and may serve as clinical biomarkers. Conclusions These results demonstrate the utility of these techniques for establishing proteomic survival signatures and for use as a discovery tool to identify candidate biomarkers for survival. This is the first clinical application of a high-throughput, large-scale LC-MS-based quantitative plasma proteomic approach for biomarker discovery for the prediction of patient outcome following burn, trauma or critical illness. PMID:23507713

  15. Invited review: Proteomics of milk and bacteria used in fermented dairy products: from qualitative to quantitative advances.

    PubMed

    Gagnaire, V; Jardin, J; Jan, G; Lortal, S

    2009-03-01

    Proteomics is a powerful tool that can simultaneously analyze several hundred proteins in complex mixtures, either through the use of high-resolution 2-dimensional gel electrophoresis or by mono- and multi-dimensional liquid chromatography coupled with mass spectrometry. Since the last review in 2005, proteomics has mainly been applied to describe minor proteins in the bovine milk fat globule membrane and soluble proteins in human colostrum. At least 130 new minor proteins have been identified. These proteins play roles in cell signaling, host defense, and transport as suggested by sequence homology. Proteomic approaches have also been applied to milk of other species such as donkey, horse, and marsupial. Peptides produced in food matrices that can exhibit functional or bioactive properties have been identified as have the proteases leading to their release in situ. However, the most spectacular proteomic development has been in the field of bacteria used in dairy products. Proteomics has resulted in the establishment of reference maps to detect strain-to-strain variations and to elucidate the mechanisms of in vitro and in vivo adaptation to environmental conditions. Proteomic analysis of bacteria entrapped in cheese has been achieved and revealed which predominant metabolic pathways are active depending on the strain. Proteomic approaches are often evoked as time-consuming procedures that provide a list of identified proteins without efficient quantification of each one. New quantitative proteomic methods have emerged and the most promising ones and their application to dairy products and bacteria will be presented.

  16. Differential Proteomics of Helicobacter pylori Associated with Autoimmune Atrophic Gastritis

    PubMed Central

    Repetto, Ombretta; Zanussi, Stefania; Casarotto, Mariateresa; Canzonieri, Vincenzo; De Paoli, Paolo; Cannizzaro, Renato; De Re, Valli

    2014-01-01

    Atrophic autoimmune gastritis (AAG) is a condition of chronic inflammation and atrophy of stomach mucosa, for which development can be partially triggered by the bacterial pathogen Helicobacter pylori (HP). HP can cause a variety of gastric diseases, such as duodenal ulcer (DU) or gastric cancer (GC). In this study, a comparative proteomic approach was used by two-dimensional fluorescence difference gel electrophoresis (DIGE) to identify differentially expressed proteins of HP strains isolated from patients with AAG, to identify markers of HP strain associated with AAG. Proteome profiles of HP isolated from GC or DU were used as a reference to compare proteomic levels. Proteomics analyses revealed 27 differentially expressed spots in AAG-associated HP in comparison with GC, whereas only 9 differential spots were found in AAG-associated HP profiles compared with DU. Proteins were identified after matrix-assisted laser desorption ionization (MALDI)-TOF and peptide mass fingerprinting. Some AAG-HP differential proteins were common between DU- and GC-HP (peroxiredoxin, heat shock protein 70 [HSP70], adenosine 5′-triphosphate [ATP] synthase subunit α, flagellin A). Our results presented here may suggest that comparative proteomes of HP isolated from AAG and DU share more common protein expression than GC and provide subsets of putative AAG-specific upregulated or downregulated proteins that could be proposed as putative markers of AAG-associated HP. Other comparative studies by two-dimensional maps integrated with functional genomics of candidate proteins will undoubtedly contribute to better decipher the biology of AAG-associated HP strains. PMID:24395566

  17. Proteomic Analysis of Menstrual Blood*

    PubMed Central

    Yang, Heyi; Zhou, Bo; Prinz, Mechthild; Siegel, Donald

    2012-01-01

    Menstruation is the expulsion of the endometrial lining of the uterus following a nearly month long preparation for embryo implantation and pregnancy. Increasingly, the health of the endometrium is being recognized as a critical factor in female fertility, and proteomes and transcriptomes from endometrial biopsies at different stages of the menstrual cycle have been studied for both diagnostic and therapeutic purposes (1 Kao, L. C., et al. 2003 Endocrinology 144, 2870–2881; Strowitzki, Tet al. 2006 Hum. Reprod. Update 12, 617–630; DeSouza, L., et al. 2005 Proteomics 5, 270–281). Disorders of the uterus ranging from benign to malignant tumors, as well as endometriosis, can cause abnormal menstrual bleeding and are frequently diagnosed through endometrial biopsy (Strowitzki, Tet al. 2006 Hum. Reprod. Update 12, 617–630; Ferenczy, A. 2003 Maturitas 45, 1–14). Yet the proteome of menstrual blood, an easily available noninvasive source of endometrial tissue, has yet to be examined for possible causes or diagnoses of infertility or endometrial pathology. This study employed five different methods to define the menstrual blood proteome. A total of 1061 proteins were identified, 361 were found by at least two methods and 678 were identified by at least two peptides. When the menstrual blood proteome was compared with those of circulating blood (1774 proteins) and vaginal fluid (823 proteins), 385 proteins were found unique to menstrual blood. Gene ontology analysis and evaluation of these specific menstrual blood proteins identified pathways consistent with the processes of the normal endometrial cycle. Several of the proteins unique to menstrual blood suggest that extramedullary uterine hematopoiesis or parenchymal hemoglobin synthesis may be occurring in late endometrial tissue. The establishment of a normal menstrual blood proteome is necessary for the evaluation of its usefulness as a diagnostic tool for infertility and uterine pathologies. Identification of

  18. Advances of Proteomic Sciences in Dentistry

    PubMed Central

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

    2016-01-01

    Applications of proteomics tools revolutionized various biomedical disciplines such as genetics, molecular biology, medicine, and dentistry. The aim of this review is to highlight the major milestones in proteomics in dentistry during the last fifteen years. Human oral cavity contains hard and soft tissues and various biofluids including saliva and crevicular fluid. Proteomics has brought revolution in dentistry by helping in the early diagnosis of various diseases identified by the detection of numerous biomarkers present in the oral fluids. This paper covers the role of proteomics tools for the analysis of oral tissues. In addition, dental materials proteomics and their future directions are discussed. PMID:27187379

  19. Microbial proteomics: the quiet revolution

    SciTech Connect

    Seraphin, Bertrand; Hettich, Robert {Bob} L

    2012-01-01

    Technological developments in DNA sequencing and their application to study thousands of microbial genomes or even microbial ecosystems still today often make the headlines of general newspapers and scientific journals. These revolutionary changes are hiding another revolution that is unfolding more quietly in the background: the development of microbial proteomics to study genome expression products. It is important to recognize that while DNA sequencing reveals extensive details about the genomic potential of an organism or community, proteomic measurements reveal the functional gene products that are present and operational under specific environmental conditions, and thus perhaps better characterize the critical biomolecules that execute the life processes (enzymes, signaling, structural factors, etc.).

  20. Liver proteomics in progressive alcoholic steatosis

    SciTech Connect

    Fernando, Harshica; Wiktorowicz, John E.; Soman, Kizhake V.; Kaphalia, Bhupendra S.; Khan, M. Firoze; Shakeel Ansari, G.A.

    2013-02-01

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

  1. Wheat proteomics: proteome modulation and abiotic stress acclimation

    PubMed Central

    Komatsu, Setsuko; Kamal, Abu H. M.; Hossain, Zahed

    2014-01-01

    Cellular mechanisms of stress sensing and signaling represent the initial plant responses to adverse conditions. The development of high-throughput “Omics” techniques has initiated a new era of the study of plant molecular strategies for adapting to environmental changes. However, the elucidation of stress adaptation mechanisms in plants requires the accurate isolation and characterization of stress-responsive proteins. Because the functional part of the genome, namely the proteins and their post-translational modifications, are critical for plant stress responses, proteomic studies provide comprehensive information about the fine-tuning of cellular pathways that primarily involved in stress mitigation. This review summarizes the major proteomic findings related to alterations in the wheat proteomic profile in response to abiotic stresses. Moreover, the strengths and weaknesses of different sample preparation techniques, including subcellular protein extraction protocols, are discussed in detail. The continued development of proteomic approaches in combination with rapidly evolving bioinformatics tools and interactive databases will facilitate understanding of the plant mechanisms underlying stress tolerance. PMID:25538718

  2. Proteomics Funding Opportunity - Office of Cancer Clinical Proteomics Research

    Cancer.gov

    To expand the understanding of how cells sense and respond to changes in their physical environment, the NCI is seeking to perform proteomic assays on the panel of cell lines grown on a variety of substrates. These assays will provide insight into changes in protein levels or phosphorylation changes that could reflect the activity of mechano-transduction pathways.

  3. Fundamentals of Plasma Physics

    NASA Astrophysics Data System (ADS)

    Bellan, Paul M.

    2008-07-01

    Preface; 1. Basic concepts; 2. The Vlasov, two-fluid, and MHD models of plasma dynamics; 3. Motion of a single plasma particle; 4. Elementary plasma waves; 5. Streaming instabilities and the Landau problem; 6. Cold plasma waves in a magnetized plasma; 7. Waves in inhomogeneous plasmas and wave energy relations; 8. Vlasov theory of warm electrostatic waves in a magnetized plasma; 9. MHD equilibria; 10. Stability of static MHD equilibria; 11. Magnetic helicity interpreted and Woltjer-Taylor relaxation; 12. Magnetic reconnection; 13. Fokker-Planck theory of collisions; 14. Wave-particle nonlinearities; 15. Wave-wave nonlinearities; 16. Non-neutral plasmas; 17. Dusty plasmas; Appendix A. Intuitive method for vector calculus identities; Appendix B. Vector calculus in orthogonal curvilinear coordinates; Appendix C. Frequently used physical constants and formulae; Bibliography; References; Index.

  4. Trypsin-catalyzed oxygen-18 labeling for quantitative proteomics

    SciTech Connect

    Qian, Weijun; Petritis, Brianne O.; Nicora, Carrie D.; Smith, Richard D.

    2011-07-01

    Stable isotope labeling based on relative peptide/protein abundance measurements is commonly applied for quantitative proteomics. Recently, trypsin-catalyzed oxygen-18 labeling has grown in popularity due to its simplicity, cost-effectiveness, and its ability to universally label peptides with high sample recovery. In (18)O labeling, both C-terminal carboxyl group atoms of tryptic peptides can be enzymatically exchanged with (18)O, thus providing the labeled peptide with a 4 Da mass shift from the (16)O-labeled sample. Peptide (18)O labeling is ideally suited for generating a labeled "universal" reference sample used for obtaining accurate and reproducible quantitative measurements across large number of samples in quantitative discovery proteomics.

  5. Proteomic analysis of the schistosome tegument and its surface membranes.

    PubMed

    Braschi, Simon; Borges, William Castro; Wilson, R Alan

    2006-09-01

    The tegument surface of the adult schistosome, bounded by a normal plasma membrane overlain by a secreted membranocalyx, holds the key to understanding how schistosomes evade host immune responses. Recent advances in mass spectrometry (MS), and the sequencing of the Schistosoma mansoni transcriptome/genome, have facilitated schistosome proteomics. We detached the tegument from the worm body and enriched its surface membranes by differential extraction, before subjecting the preparation to liquid chromatography-based proteomics to identify its constituents. The most exposed proteins on live worms were labelled with impearmeant biotinylation reagents, and we also developed methods to isolate the membranocalyx for analysis. We identified transporters for sugars, amino acids, inorganic ions and water, which confirm the importance of the tegument plasma membrane in nutrient acquisition and solute balance. Enzymes, including phosphohydrolases, esterases and carbonic anhydrase were located with their catalytic domains external to the plasma membrane, while five tetraspanins, annexin and dysferlin were implicated in membrane architecture. In contrast, few parasite proteins could be assigned to the membranocalyx but mouse immune response proteins, including three immunoglobulins and two complement factors, were detected, plus host membrane proteins such as CD44, integrin and a complement regulatory protein, testifying to the acquisitive properties of the secreted bilayer. PMID:17308771

  6. Determination of hydride forming elements (As, Sb, Se, Sn) and Hg in environmental reference materials as acid slurries by on-line hydride generation inductively coupled plasma mass spectrometry

    NASA Astrophysics Data System (ADS)

    Ribeiro, Anderson Schwingel; Vieira, Mariana Antunes; Curtius, Adilson José

    2004-02-01

    A method for the determination of As, Hg, Sb, Se and Sn in environmental and in geological reference materials, as acidified slurries, by flow injection (FI) coupled to a hydride generation system (HG) and detection by inductively coupled plasma mass spectrometry (ICP-MS) is proposed. The HG unit has a gas liquid separator and a drying unit for the generated vapor. The slurries were prepared by two procedures. Approximately 50 mg of the reference material, ground to a particle size ≤50 μm, was mixed with acid solutions in an ultrasonic bath. In Procedure A, the medium was a hydrochloric acid solution while in Procedure B, the medium was aqua regia plus a hydrochloric acid solution. The conditions for the slurry formation and the instrumental parameters were optimized. Harsh conditions were used in the slurry preparation in order to reduce the hydride forming analytes to their lower oxidation states, As (III), Se(IV), Sb(III) and Sn(II), before reacting with sodium tetrahydroborate. To test the accuracy, 10 certified reference materials were analyzed (four sediments, three coals, one coal fly ash and two sewage sludges), with the analyte concentrations mostly in the μg g -1 level. Good agreements with the certified values were obtained for Hg, Sb and Sn in the sediments using Procedure A and calibration against aqueous standard solutions. Using Procedure B, good results were obtained for Hg, Se and Sn in the sediment samples, for Se in the coal and coal fly ash samples and for Hg in the sewage sludge samples, also using external calibration with aqueous standard solutions. For As in sediments, coals and coal fly ash, Procedure B and the analyte addition calibration was required, indicating matrix effects. The relative standard deviations were lower than 5%, demonstrating a good precision for slurry analysis. The limits of quantification (10 times the standard deviation; n=10), in the samples, in ng g -1, were: 20 for As, 60 for Hg, 80 for Sb, 200 for Se and 90

  7. Comparative proteome analysis across non-small cell lung cancer cell lines.

    PubMed

    Grundner-Culemann, Kathrin; Dybowski, J Nikolaj; Klammer, Martin; Tebbe, Andreas; Schaab, Christoph; Daub, Henrik

    2016-01-01

    Non-small cell lung cancer (NSCLC) cell lines are widely used model systems to study molecular aspects of lung cancer. Comparative and in-depth proteome expression data across many NSCLC cell lines has not been generated yet, but would be of utility for the investigation of candidate targets and markers in oncogenesis. We employed a SILAC reference approach to perform replicate proteome quantifications across 23 distinct NSCLC cell lines. On average, close to 4000 distinct proteins were identified and quantified per cell line. These included many known targets and diagnostic markers, indicating that our proteome expression data represents a useful resource for NSCLC pre-clinical research. To assess proteome diversity within the NSCLC cell line panel, we performed hierarchical clustering and principal component analysis of proteome expression data. Our results indicate that general proteome diversity among NSCLC cell lines supersedes potential effects common to K-Ras or epidermal growth factor receptor (EGFR) oncoprotein expression. However, we observed partial segregation of EGFR or KRAS mutant cell lines for certain principal components, which reflected biological differences according to gene ontology enrichment analyses. Moreover, statistical analysis revealed several proteins that were significantly overexpressed in KRAS or EGFR mutant cell lines. PMID:26361996

  8. Expansion of the ion library for mining SWATH-MS data through fractionation proteomics.

    PubMed

    Zi, Jin; Zhang, Shenyan; Zhou, Ruo; Zhou, Baojin; Xu, Shaohang; Hou, Guixue; Tan, Fengji; Wen, Bo; Wang, Quanhui; Lin, Liang; Liu, Siqi

    2014-08-01

    The strategy of sequential window acquisition of all theoretical fragment ion spectra (SWATH) is emerging in the field of label-free proteomics. A critical consideration for the processing of SWATH data is the quality of the ion library (or mass spectrometric reference map). As the availability of open spectral libraries that can be used to process SWATH data is limited, most users currently create their libraries in-house. Herein, we propose an approach to construct an expanded ion library using the data-dependent acquisition (DDA) data generated by fractionation proteomics. We identified three critical elements for achieving a satisfactory ion library during the iterative process of our ion library expansion, including a correction of the retention times (RTs) gained from fractionation proteomics, appropriate integrations of the fractionated proteomics into an ion library, and assessments of the impact of the expanded ion libraries to data mining in SWATH. Using a bacterial lysate as an evaluation material, we employed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to fractionate the lysate proteins and constructed the expanded ion library using the fractionation proteomics data. Compared with the ion library built from the unfractionated proteomics, approximately 20% more peptides were extracted from the expanded ion library. The extracted peptides, moreover, were acceptable for further quantitative analysis.

  9. The speciation of the proteome

    PubMed Central

    Jungblut, Peter R; Holzhütter, Hermann G; Apweiler, Rolf; Schlüter, Hartmut

    2008-01-01

    Introduction In proteomics a paradox situation developed in the last years. At one side it is basic knowledge that proteins are post-translationally modified and occur in different isoforms. At the other side the protein expression concept disclaims post-translational modifications by connecting protein names directly with function. Discussion Optimal proteome coverage is today reached by bottom-up liquid chromatography/mass spectrometry. But quantification at the peptide level in shotgun or bottom-up approaches by liquid chromatography and mass spectrometry is completely ignoring that a special peptide may exist in an unmodified form and in several-fold modified forms. The acceptance of the protein species concept is a basic prerequisite for meaningful quantitative analyses in functional proteomics. In discovery approaches only top-down analyses, separating the protein species before digestion, identification and quantification by two-dimensional gel electrophoresis or protein liquid chromatography, allow the correlation between changes of a biological situation and function. Conclusion To obtain biological relevant information kinetics and systems biology have to be performed at the protein species level, which is the major challenge in proteomics today. PMID:18638390

  10. Periodontal Proteomics: Wonders Never Cease!

    PubMed Central

    Grover, Harpreet Singh; Kapoor, Shalini; Saksena, Neha

    2013-01-01

    Proteins are vital parts of living organisms, as they are integral components of the physiological metabolic pathways of cells. Periodontal tissues comprise multicompartmental groups of interacting cells and matrices that provide continuous support, attachment, proprioception, and physical protection for the teeth. The proteome map, that is, complete catalogue of the matrix and cellular proteins expressed in alveolar bone, cementum, periodontal ligament, and gingiva, is to be explored for more in-depth understanding of periodontium. The ongoing research to understand the signalling pathways that allow cells to divide, differentiate, and die in controlled manner has brought us to the era of proteomics. Proteomics is defined as the study of all proteins including their relative abundance, distribution, posttranslational modifications, functions, and interactions with other macromolecules, in a given cell or organism within a given environment and at a specific stage in the cell cycle. Its application to periodontal science can be used to monitor health status, disease onset, treatment response, and outcome. Proteomics can offer answers to critical, unresolved questions such as the biological basis for the heterogeneity in gingival, alveolar bone, and cemental cell populations. PMID:24490073

  11. The proteome of human retina

    PubMed Central

    Zhang, Pingbo; Dufresne, Craig; Turner, Randi; Ferri, Sara; Venkatraman, Vidya; Karani, Rabia; Lutty, Gerard A.; Van Eyk, Jennifer E.; Semba, Richard D.

    2014-01-01

    The retina is a delicate tissue that detects light, converts photochemical energy into neural signals, and transmits the signals to the visual cortex of the brain. A detailed protein inventory of the proteome of the normal human eye may provide a foundation for new investigations into both the physiology of the retina and the pathophysiology of retinal diseases. To provide an inventory, proteins were extracted from five retinas of normal eyes and fractionated using SDS-PAGE. After in-gel digestion, peptides were analyzed in duplicate using LC-MS/MS on an Orbitrap Elite mass spectrometer. A total of 3,436 non-redundant proteins were identified in the human retina, including 20 unambiguous protein isoforms, of which 8 have not previously been demonstrated to exist at the protein level. The proteins identified in the retina included most of the enzymes involved in the visual cycle and retinoid metabolism. One hundred and fifty-eight proteins that have been associated with age-related macular degeneration were identified in the retina. The MS proteome database of the human retina may serve as a valuable resource for future investigations of retinal biology and disease. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD001242. PMID:25407473

  12. Proteomics of foodborne bacterial pathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This chapter focuses on recent research on foodborne bacterial pathogens that use mass spectrometry-based proteomic techniques as well as protein microarrays. Mass spectrometry ionization techniques (e.g. electrospray ionization and matrix-assisted laser desorption/ionization), analyzers (e.g. ion ...

  13. The Size of the Human Proteome: The Width and Depth

    PubMed Central

    Ponomarenko, Elena A.; Poverennaya, Ekaterina V.; Ilgisonis, Ekaterina V.; Pyatnitskiy, Mikhail A.; Kopylov, Arthur T.; Zgoda, Victor G.; Lisitsa, Andrey V.; Archakov, Alexander I.

    2016-01-01

    This work discusses bioinformatics and experimental approaches to explore the human proteome, a constellation of proteins expressed in different tissues and organs. As the human proteome is not a static entity, it seems necessary to estimate the number of different protein species (proteoforms) and measure the number of copies of the same protein in a specific tissue. Here, meta-analysis of neXtProt knowledge base is proposed for theoretical prediction of the number of different proteoforms that arise from alternative splicing (AS), single amino acid polymorphisms (SAPs), and posttranslational modifications (PTMs). Three possible cases are considered: (1) PTMs and SAPs appear exclusively in the canonical sequences of proteins, but not in splice variants; (2) PTMs and SAPs can occur in both proteins encoded by canonical sequences and in splice variants; (3) all modification types (AS, SAP, and PTM) occur as independent events. Experimental validation of proteoforms is limited by the analytical sensitivity of proteomic technology. A bell-shaped distribution histogram was generated for proteins encoded by a single chromosome, with the estimation of copy numbers in plasma, liver, and HepG2 cell line. The proposed metabioinformatics approaches can be used for estimation of the number of different proteoforms for any group of protein-coding genes. PMID:27298622

  14. Recent advances in yeast organelle and membrane proteomics.

    PubMed

    Premsler, Thomas; Zahedi, René Peiman; Lewandrowski, Urs; Sickmann, Albert

    2009-10-01

    Yeast proteome research comprises two different aspects: with respect to systemic fungal infections (fungemias), invasive candidiasis, for instance by Candida albicans, is among the most common causes of morbidity and mortality particularly in the expanding population of immunocompromised patients, which rises a high medical and pharmaceutical interest in this facultative pathogenic organism. Apart from its clinical relevance, yeast research moreover provides an indispensable source of knowledge regarding fundamental biochemical processes of eukaryotic cells. In this context, the budding yeast Saccharomyces cerevisiae is, in addition to its multiple industrial applications, one of the most extensively used microorganisms and serves as the best understood eukaryotic model system so far. Consequently, numerous studies have focused on gaining insight into the yeast proteome, with protein MS providing a very efficient technology to cope with this task since it enables both protein identification and differential quantification of cellular material. In this review we present an overview of recent advances in yeast organelle and membrane proteomics focusing on the cell wall, plasma membrane, mitochondria and vacuole.

  15. A Combined Metabolomic and Proteomic Analysis of Gestational Diabetes Mellitus.

    PubMed

    Hajduk, Joanna; Klupczynska, Agnieszka; Dereziński, Paweł; Matysiak, Jan; Kokot, Piotr; Nowak, Dorota M; Gajęcka, Marzena; Nowak-Markwitz, Ewa; Kokot, Zenon J

    2015-12-16

    The aim of this pilot study was to apply a novel combined metabolomic and proteomic approach in analysis of gestational diabetes mellitus. The investigation was performed with plasma samples derived from pregnant women with diagnosed gestational diabetes mellitus (n = 18) and a matched control group (n = 13). The mass spectrometry-based analyses allowed to determine 42 free amino acids and low molecular-weight peptide profiles. Different expressions of several peptides and altered amino acid profiles were observed in the analyzed groups. The combination of proteomic and metabolomic data allowed obtaining the model with a high discriminatory power, where amino acids ethanolamine, L-citrulline, L-asparagine, and peptide ions with m/z 1488.59; 4111.89 and 2913.15 had the highest contribution to the model. The sensitivity (94.44%) and specificity (84.62%), as well as the total group membership classification value (90.32%) calculated from the post hoc classification matrix of a joint model were the highest when compared with a single analysis of either amino acid levels or peptide ion intensities. The obtained results indicated a high potential of integration of proteomic and metabolomics analysis regardless the sample size. This promising approach together with clinical evaluation of the subjects can also be used in the study of other diseases.

  16. Exploring analytical proteomics platforms toward the definition of human cardiac stem cells receptome.

    PubMed

    Gomes-Alves, Patrícia; Serra, Margarida; Brito, Catarina; R-Borlado, Luis; López, Juan A; Vázquez, Jesús; Carrondo, Manuel J T; Bernad, António; Alves, Paula M

    2015-04-01

    Human cardiac stem cells (hCSC) express a portfolio of plasma membrane receptors that are involved in the regulatory auto/paracrine feedback loop mechanism of activation of these cells, and consequently contribute to myocardial regeneration. In order to attain a comprehensive description of hCSC receptome and overcoming the inability demonstrated by other technologies applied in receptor identification, mainly due to the transmembrane nature, high hydrophobic character and relative low concentration of these proteins, we have exploited and improved a proteomics workflow. This approach was based on the enrichment of hCSC plasma membrane fraction and addition of prefractionation steps prior to MS analysis. More than 100 plasma membrane receptors were identified. The data reported herein constitute a valuable source of information to further understand cardiac stem cells activation mechanisms and the subsequent cardiac repair process. All MS data have been deposited in the ProteomeXchange with identifier PXD001117 (http://proteomecentral.proteomexchange.org/dataset/PXD001117).

  17. BioAfrica's HIV-1 proteomics resource: combining protein data with bioinformatics tools.

    PubMed

    Doherty, Ryan S; De Oliveira, Tulio; Seebregts, Chris; Danaviah, Sivapragashini; Gordon, Michelle; Cassol, Sharon

    2005-01-01

    Most Internet online resources for investigating HIV biology contain either bioinformatics tools, protein information or sequence data. The objective of this study was to develop a comprehensive online proteomics resource that integrates bioinformatics with the latest information on HIV-1 protein structure, gene expression, post-transcriptional/post-translational modification, functional activity, and protein-macromolecule interactions. The BioAfrica HIV-1 Proteomics Resource http://bioafrica.mrc.ac.za/proteomics/index.html is a website that contains detailed information about the HIV-1 proteome and protease cleavage sites, as well as data-mining tools that can be used to manipulate and query protein sequence data, a BLAST tool for initiating structural analyses of HIV-1 proteins, and a proteomics tools directory. The Proteome section contains extensive data on each of 19 HIV-1 proteins, including their functional properties, a sample analysis of HIV-1HXB2, structural models and links to other online resources. The HIV-1 Protease Cleavage Sites section provides information on the position, subtype variation and genetic evolution of Gag, Gag-Pol and Nef cleavage sites. The HIV-1 Protein Data-mining Tool includes a set of 27 group M (subtypes A through K) reference sequences that can be used to assess the influence of genetic variation on immunological and functional domains of the protein. The BLAST Structure Tool identifies proteins with similar, experimentally determined topologies, and the Tools Directory provides a categorized list of websites and relevant software programs. This combined database and software repository is designed to facilitate the capture, retrieval and analysis of HIV-1 protein data, and to convert it into clinically useful information relating to the pathogenesis, transmission and therapeutic response of different HIV-1 variants. The HIV-1 Proteomics Resource is readily accessible through the BioAfrica website at: http://bioafrica.mrc.ac.za/proteomics

  18. CPTAC Releases Largest-Ever Breast Cancer Proteome Dataset - Office of Cancer Clinical Proteomics Research

    Cancer.gov

    National Cancer Institute (NCI) Clinical Proteomic Tumor Analysis Consortium (CPTAC) scientists have released a dataset of proteins and phophorylated phosphopeptides identified through deep proteomic and phosphoproteomic analysis of breast tumor samples, previously genomically analyzed by The Cancer Genome Atlas (TCGA).

  19. NCI Launches Proteomics Assay Portal - Office of Cancer Clinical Proteomics Research

    Cancer.gov

    In a paper recently published by the journal Nature Methods, Investigators from the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (NCI-CPTAC) announced the launch of a proteomics Assay Portal for multiple reaction monitoring-mass

  20. Proteomics Data on UCSC Genome Browser - Office of Cancer Clinical Proteomics Research

    Cancer.gov

    The National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium scientists are working together with the University of California, Santa Cruz (UCSC) Genomics Institute to provide public access to cancer proteomics data.

  1. DynaProt 2D: an advanced proteomic database for dynamic online access to proteomes and two-dimensional electrophoresis gels

    PubMed Central

    Drews, Oliver; Görg, Angelika

    2005-01-01

    DynaProt 2D presents an advanced online database for dynamic access to proteomes and two-dimensional (2D) gels. The database was designed to administer complete in silico proteomes and links them with experimental proteomic data in the manner of 2D electrophoresis gels (IPG-Dalt). The 2D gels serve as reference maps in 2D gel analysis as well as tools for navigation of the database to switch between experimental and predicted data. Therefore, all identified spots in the gels are clickable and linked with summarized protein information. The protein information tables contain calculated characteristics, which are often used in proteomics, such as the molecular weight, isoelectric point, codon adaptation index, grand average of hydropathicity, etc. The design of the database permits online extension of gel data and protein attributes without knowledge of any software language. Besides navigation via 2D gels, the clear graphical user interface permits quick and intuitive searching throughout complete proteomes and supports, e.g. the search for proteins with isoelectric points within pH ranges of interest or protein classes (e.g. ribosomal proteins or transporters). The first organism implemented in the database is Lactococcus lactis. The database is available at www.wzw.tum.de/proteomik/lactis. PMID:15608266

  2. Data for chicken semen proteome and label free quantitative analyses displaying sperm quality biomarkers.

    PubMed

    Labas, Valérie; Grasseau, Isabelle; Cahier, Karine; Gargaros, Audrey; Harichaux, Grégoire; Teixeira-Gomes, Ana-Paula; Alves, Sabine; Bourin, Marie; Gérard, Nadine; Blesbois, Elisabeth

    2014-12-01

    Understanding of biology of the avian male gamete is essential to improve the conservation of genetic resources and performances in farming. In this study, the semen proteome of the main domestic avian species (Gallus gallus) and evaluation of the molecular phenotype related to sperm quality were investigated using GeLC-MS/MS approach and label-free quantitative proteomic based on Spectral Counting (SC) and extracted ion chromatograms (XIC) methods. Here we describe in details the peptide/protein inventory of chicken ejaculated spermatozoa (SPZ) and seminal plasma (SP). We also show differential analyses of chicken semen (SPZ and corresponding SP) from 11 males demonstrating different levels of fertilizing capacity and sperm motility. The interpretation and description of these data can be found in a research article published by Labas and colleagues in the Journal of Proteomics in 2014 [1]. This is a new resource for exploring the molecular mechanisms involved in fertilizing capacity and to reveal new sets of fertility biomarkers.

  3. Recent advances in chemical proteomics: exploring the post-translational proteome.

    PubMed

    Tate, Edward W

    2008-11-01

    Identification and quantification of multiple proteins from complex mixtures is a central theme in post-genomic biology. Despite recent progress in high-throughput proteomics, proteomic analysis of post-translationally modified (PTM) proteins remains particularly challenging. This mini-review introduces the emerging field of chemical proteomics and reviews recent advances in chemical proteomic technology that are offering striking new insights into the functional biology of post-translational modification.

  4. Exploring the potential of public proteomics data

    PubMed Central

    Vaudel, Marc; Verheggen, Kenneth; Csordas, Attila; Ræder, Helge; Berven, Frode S.; Martens, Lennart; Vizcaíno, Juan A.

    2015-01-01

    In a global effort for scientific transparency, it has become feasible and good practice to share experimental data supporting novel findings. Consequently, the amount of publicly available MS‐based proteomics data has grown substantially in recent years. With some notable exceptions, this extensive material has however largely been left untouched. The time has now come for the proteomics community to utilize this potential gold mine for new discoveries, and uncover its untapped potential. In this review, we provide a brief history of the sharing of proteomics data, showing ways in which publicly available proteomics data are already being (re‐)used, and outline potential future opportunities based on four different usage types: use, reuse, reprocess, and repurpose. We thus aim to assist the proteomics community in stepping up to the challenge, and to make the most of the rapidly increasing amount of public proteomics data. PMID:26449181

  5. Proteomics and Ovarian Cancer: Integrating Proteomics Information Into Clinical Care

    PubMed Central

    Hays, John L.; Kim, Geoffrey; Giuroiu, Iulia; Kohn, Elise C.

    2010-01-01

    The power of proteomics allows unparalleled opportunity to query the molecular mechanisms of a malignant cell and the tumor microenvironment in patients with ovarian cancer and other solid tumors. This information has given us insight into the perturbations of signaling pathways within tumor cells and has aided the discovery of new drug targets for the tumor and possible prognostic indicators of outcome and disease response to therapy. Proteomics analysis of serum and ascites has also given us sources with which to discover possible early markers for the presence of new disease and for the progression of established cancer throughout the course of treatment. Unfortunately, this wealth of information has yielded little to date in changing the clinical care of these patients from a diagnostic, prognostic, or treatment perspective. The rational examination and translation of proteomics data in the context of past clinical trials and the design of future clinical trials must occur before we can march forward into the future of personalized medicine. PMID:20561909

  6. Proteomics and Metabolomics: Two Emerging Areas for Legume Improvement.

    PubMed

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

    2015-01-01

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

  7. Proteomics and Metabolomics: Two Emerging Areas for Legume Improvement

    PubMed Central

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

    2015-01-01

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

  8. Proteomic approaches in research of cyanobacterial photosynthesis.

    PubMed

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

    2015-10-01

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

  9. MetazSecKB: the human and animal secretome and subcellular proteome knowledgebase

    PubMed Central

    Meinken, John; Walker, Gary; Cooper, Chester R.; Min, Xiang Jia

    2015-01-01

    The subcellular location of a protein is a key factor in determining the molecular function of the protein in an organism. MetazSecKB is a secretome and subcellular proteome knowledgebase specifically designed for metazoan, i.e. human and animals. The protein sequence data, consisting of over 4 million entries with 121 species having a complete proteome, were retrieved from UniProtKB. Protein subcellular locations including secreted and 15 other subcellular locations were assigned based on either curated experimental evidence or prediction using seven computational tools. The protein or subcellular proteome data can be searched and downloaded using several different types of identifiers, gene name or keyword(s), and species. BLAST search and community annotation of subcellular locations are also supported. Our primary analysis revealed that the proteome sizes, secretome sizes and other subcellular proteome sizes vary tremendously in different animal species. The proportions of secretomes vary from 3 to 22% (average 8%) in metazoa species. The proportions of other major subcellular proteomes ranged approximately 21–43% (average 31%) in cytoplasm, 20–37% (average 30%) in nucleus, 3–19% (average 12%) as plasma membrane proteins and 3–9% (average 6%) in mitochondria. We also compared the protein families in secretomes of different primates. The Gene Ontology and protein family domain analysis of human secreted proteins revealed that these proteins play important roles in regulation of human structure development, signal transduction, immune systems and many other biological processes. Database URL: http://proteomics.ysu.edu/secretomes/animal/index.php PMID:26255309

  10. Proteomics by FTICR Mass Spectrometry: Top Down and Bottom Up

    SciTech Connect

    Bogdanov, Bogdan; Smith, Richard D.

    2005-03-31

    This review offers a broad overview of recent FTICR applications and technological developments in the field of proteomics, directed to a variety of people with different expertise and interests. Both the ''bottom-up'' (peptide level) and ''top-down'' (intact protein level) approaches will be covered and various related aspects will be discussed and illustrated with examples that are among the best available references in the literature. ''Bottom-up topics include peptide fragmentation, the AMT approach and DREAMS technology, quantitative proteomics, post-translational modifications, and special FTICR software focused on peptide and protein identification. Topics in the ''top-down'' part include various aspects of high-mass measurements, protein tandem mass spectrometry, protein confirmations, protein-protein complexes, as well as some esoteric applications that may become more practical in the coming years. Finally, examples of integrating both approaches and medical proteomics applications using FTICR will be provided, closing with an outlook of what may be coming our way sooner than later.

  11. Applied proteomics: mitochondrial proteins and effect on function.

    PubMed

    Lopez, Mary F; Melov, Simon

    2002-03-01

    The identification of a majority of the polypeptides in mitochondria would be invaluable because they play crucial and diverse roles in many cellular processes and diseases. The endogenous production of reactive oxygen species (ROS) is a major limiter of life as illustrated by studies in which the transgenic overexpression in invertebrates of catalytic antioxidant enzymes results in increased lifespans. Mitochondria have received considerable attention as a principal source---and target---of ROS. Mitochondrial oxidative stress has been implicated in heart disease including myocardial preconditioning, ischemia/reperfusion, and other pathologies. In addition, oxidative stress in the mitochondria is associated with the pathogenesis of Alzheimer's disease, Parkinson's disease, prion diseases, and amyotrophic lateral sclerosis (ALS) as well as aging itself. The rapidly emerging field of proteomics can provide powerful strategies for the characterization of mitochondrial proteins. Current approaches to mitochondrial proteomics include the creation of detailed catalogues of the protein components in a single sample or the identification of differentially expressed proteins in diseased or physiologically altered samples versus a reference control. It is clear that for any proteomics approach prefractionation of complex protein mixtures is essential to facilitate the identification of low-abundance proteins because the dynamic range of protein abundance within cells has been estimated to be as high as 10(7). The opportunities for identification of proteins directly involved in diseases associated with or caused by mitochondrial dysfunction are compelling. Future efforts will focus on linking genomic array information to actual protein levels in mitochondria. PMID:11884366

  12. Combining Capillary Electrophoresis with Mass Spectrometry for Applications in Proteomics

    SciTech Connect

    Simpson, David C.; Smith, Richard D.

    2005-04-01

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

  13. Integrative Analysis of the Mitochondrial Proteome in Yeast

    SciTech Connect

    Prokisch, Holger; Scharfe, Curt M.; Camp, David G.; Xiao, Wenzhong; David, Lior; Andreoli, Christophe; Monroe, Matthew E.; Moore, Ronald J.; Gritsenko, Marina A.; Kozany, Christian; Hixson, Kim K.; Mottaz, Heather M.; Zischka, Hans; Ueffing, Marius; Herman, Zelek S.; Davis, Ronald W.; Meitinger, Thomas; Oefner, Peter; Smith, Richard D.; Steinmetz, Lars M.

    2004-06-30

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

  14. Biospecimen 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.

  15. A Quantitative Glycomics and Proteomics Combined Purification Strategy

    PubMed Central

    Muddiman, David C.

    2016-01-01

    There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define pathways, improve treatment, understand disease, and aid biomarker discovery. N-linked glycosylation is one of the most important and robust post-translational modifications on proteins and regulates critical cell functions such as signaling, adhesion, and enzymatic function. Analytical techniques to purify and analyze N-glycans have remained relatively static over the last decade. While accurate and effective, they commonly require significant expertise and resources. Though some high-throughput purification schemes have been developed, they have yet to find widespread adoption and often rely on the enrichment of glycopeptides. One promising method, developed by Thomas-Oates et al., filter aided N-glycan separation (FANGS), was qualitatively demonstrated on tissues. Herein, we adapted FANGS to plasma and coupled it to the individuality normalization when labeling with glycan hydrazide tags strategy in order to achieve accurate relative quantification by liquid chromatography mass spectrometry and enhanced electrospray ionization. Furthermore, we designed new functionality to the protocol by achieving tandem, shotgun proteomics and glycosylation site analysis on hen plasma. We showed that N-glycans purified on filter and derivatized by hydrophobic hydrazide tags were comparable in terms of abundance and class to those by solid phase extraction (SPE); the latter is considered a gold standard in the field. Importantly, the variability in the two protocols was not statistically different. Proteomic data that was collected in-line with glycomic data had the same depth compared to a standard trypsin digest. Peptide deamidation is minimized in the protocol, limiting non-specific deamidation detected at glycosylation motifs. This allowed for direct glycosylation site analysis, though the protocol can accommodate 18O site

  16. Proteomics of Leaf Tissues from Populus

    SciTech Connect

    Hurst, Gregory {Greg} B; Yang, Xiaohan; Tschaplinski, Timothy J; Tuskan, Gerald A; Lankford, Patricia K; Shah, Manesh B; Jawdy, Sara; Gunter, Lee E; Engle, Nancy L

    2010-01-01

    Trees of the genus Populus are farmed commercially for wood and fiber, and are a potential bioenergy crop. As a scientific model organism, P. trichocarpa was the first forest tree for which the genome sequence has been determined. Knowledge of the Populus proteome will provide a deeper understanding of gene expression patterns in various tissues of the plant. To build on our previous profile of the proteome of xylem tissue in Populus (Kalluri et al., Proteomics 2009, 9, 4871), we are currently developing methods for studying the proteome of Populus leaves.

  17. Informatics solutions for high-throughput proteomics.

    PubMed

    Topaloglou, Thodoros

    2006-06-01

    The success of mass-spectrometry-based proteomics as a method for analyzing proteins in biological samples is accompanied by challenges owning to demands for increased throughput. These challenges arise from the vast volume of data generated by proteomics experiments combined with the heterogeneity in data formats, processing methods, software tools and databases that are involved in the translation of spectral data into relevant and actionable information for scientists. Informatics aims to provide answers to these challenges by transferring existing solutions from information management to proteomics and/or by generating novel computational methods for automation of proteomics data processing.

  18. Quantitative Proteomic Analysis of the Human Nucleolus.

    PubMed

    Bensaddek, Dalila; Nicolas, Armel; Lamond, Angus I

    2016-01-01

    Recent years have witnessed spectacular progress in the field of mass spectrometry (MS)-based quantitative proteomics, including advances in instrumentation, chromatography, sample preparation methods, and experimental design for multidimensional analyses. It is now possible not only to identify most of the protein components of a cell proteome in a single experiment, but also to describe additional proteome dimensions, such as protein turnover rates, posttranslational modifications, and subcellular localization. Furthermore, by comparing the proteome at different time points, it is possible to create a "time-lapse" view of proteome dynamics. By combining high-throughput quantitative proteomics with detailed subcellular fractionation protocols and data analysis techniques it is also now possible to characterize in detail the proteomes of specific subcellular organelles, providing important insights into cell regulatory mechanisms and physiological responses. In this chapter we present a reliable workflow and protocol for MS-based analysis and quantitation of the proteome of nucleoli isolated from human cells. The protocol presented is based on a SILAC analysis of human MCF10A-Src-ER cells with analysis performed on a Q-Exactive Plus Orbitrap MS instrument (Thermo Fisher Scientific). The subsequent chapter describes how to process the resulting raw MS files from this experiment using MaxQuant software and data analysis procedures to evaluate the nucleolar proteome using customized R scripts. PMID:27576725

  19. Proteomic biomarkers in lung cancer.

    PubMed

    Pastor, M D; Nogal, A; Molina-Pinelo, S; Carnero, A; Paz-Ares, L

    2013-09-01

    The correct understanding of tumour development relies on the comprehensive study of proteins. They are the main orchestrators of vital processes, such as signalling pathways, which drive the carcinogenic process. Proteomic technologies can be applied to cancer research to detect differential protein expression and to assess different responses to treatment. Lung cancer is the number one cause of cancer-related death in the world. Mostly diagnosed at late stages of the disease, lung cancer has one of the lowest 5-year survival rates at 15 %. The use of different proteomic techniques such as two-dimensional gel electrophoresis (2D-PAGE), isotope labelling (ICAT, SILAC, iTRAQ) and mass spectrometry may yield new knowledge on the underlying biology of lung cancer and also allow the development of new early detection tests and the identification of changes in the cancer protein network that are associated with prognosis and drug resistance. PMID:23606351

  20. Brucella proteomes--a review.

    PubMed

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

    2002-12-20

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

  1. Human Saliva Proteome and Transcriptome

    PubMed Central

    Hu, S.; Li, Y.; Wang, J.; Xie, Y.; Tjon, K.; Wolinsky, L.; Loo, R.R.O.; Loo, J.A.; Wong, D.T.

    2007-01-01

    This paper tests the hypothesis that salivary proteins and their counterpart mRNAs co-exist in human whole saliva. Global profiling of human saliva proteomes and transcriptomes by mass spectrometry (MS) and expression microarray technologies, respectively, revealed many similarities between saliva proteins and mRNAs. Of the function-known proteins identified in saliva, from 61 to 70% were also found present as mRNA transcripts. For genes not detected at both protein and mRNA levels, we made further efforts to determine if the counterpart is present. Of 19 selected genes detected only at the protein level, the mRNAs of 13 (68%) genes were found in saliva by RT-PCR. In contrast, of many mRNAs detected only by microarrays, their protein products were found in saliva, as reported previously by other investigators. The saliva transcriptome may provide preliminary insights into the boundary of the saliva proteome. PMID:17122167

  2. Activating stimuli induce platelet microRNA modulation and proteome reorganisation.

    PubMed

    Cimmino, Giovanni; Tarallo, Roberta; Nassa, Giovanni; De Filippo, Maria Rosaria; Giurato, Giorgio; Ravo, Maria; Rizzo, Francesca; Conte, Stefano; Pellegrino, Grazia; Cirillo, Plinio; Calabro, Paolo; Öhman, Tiina; Nyman, Tuula A; Weisz, Alessandro; Golino, Paolo

    2015-07-01

    Platelets carry megakaryocyte-derived mRNAs whose translation efficiency before and during activation is not known, although this can greatly affect platelet functions, both under basal conditions and in response to physiological and pathological stimuli, such as those involved in acute coronary syndromes. Aim of the present study was to determine whether changes in microRNA (miRNA) expression occur in response to activating stimuli and whether this affects activity and composition of platelet transcriptome and proteome. Purified platelet-rich plasmas from healthy volunteers were collected and activated with ADP, collagen, or thrombin receptor activating peptide. Transcriptome analysis by RNA-Seq revealed that platelet transcriptome remained largely unaffected within the first 2 hours of stimulation. In contrast, quantitative proteomics showed that almost half of > 700 proteins quantified were modulated under the same conditions. Global miRNA analysis indicated that reorganisation of platelet proteome occurring during activation reflected changes in mature miRNA expression, which therefore, appears to be the main driver of the observed discrepancy between transcriptome and proteome changes. Platelet functions significantly affected by modulated miRNAs include, among others, the integrin/cytoskeletal, coagulation and inflammatory-immune response pathways. These results demonstrate a significant reprogramming of the platelet miRNome during activation, with consequent significant changes in platelet proteome and provide for the first time substantial evidence that fine-tuning of resident mRNA translation by miRNAs is a key event in platelet pathophysiology. PMID:25903651

  3. Rapid fabrication of glass/PDMS hybrid µIMER for high throughput membrane proteomics.

    PubMed

    Pereira-Medrano, Ana G; Forster, Simon; Fowler, Gregory J S; McArthur, Sally L; Wright, Phillip C

    2010-12-21

    Mass spectrometry (MS) based proteomics has brought a radical approach to systems biology, offering a platform to study complex biological functions. However, key proteomic technical challenges remain, mainly the inability to characterise the complete proteome of a cell due to the thousands of diverse, complex proteins expressed at an extremely wide concentration range. Currently, high throughput and efficient techniques to unambiguously identify and quantify proteins on a proteome-wide scale are in demand. Miniaturised analytical systems placed upstream of MS help us to attain these goals. One time-consuming step in traditional techniques is the in-solution digestion of proteins (4-20 h). This also has other drawbacks, including enzyme autoproteolysis, low efficiency, and manual operation. Furthermore, the identification of α-helical membrane proteins has remained a challenge due to their high hydrophobicity and lack of trypsin cleavage targets in transmembrane helices. We demonstrate a new rapidly produced glass/PDMS micro Immobilised Enzyme Reactor (µIMER) with enzymes covalently immobilised onto polyacrylic acid plasma-modified surfaces for the purpose of rapidly (as low as 30 s) generating peptides suitable for MS analysis. This µIMER also allows, for the first time, rapid digestion of insoluble proteins. Membrane protein identification through this method was achieved after just 4 min digestion time, up to 9-fold faster than either dual-stage in-solution digestion approaches or other commonly used bacterial membrane proteomic workflows.

  4. International reference standards in coagulation.

    PubMed

    Raut, Sanj; Hubbard, Anthony R

    2010-07-01

    Measurement of coagulation factor activity using absolute physico-chemical techniques is not possible and estimation therefore relies on comparative bioassay relative to a reference standard with a known or assigned potency. However the inherent variability of locally prepared and calibrated reference standards can give rise to poor agreement between laboratories and methods. Harmonisation of measurement between laboratories at the international level relies on the availability of a common source of calibration for local reference standards and this is provided by the World Health Organization (WHO) International Standards which define the International Unit for the analyte. This article describes the principles, practices and problems of biological standardisation and the development and use of reference standards for assays of coagulation factors, with particular emphasis on WHO International Standards for both concentrates and plasma.

  5. A visual approach to proteomics.

    PubMed

    Nickell, Stephan; Kofler, Christine; Leis, Andrew P; Baumeister, Wolfgang

    2006-03-01

    Cryo-electron tomography is an emerging imaging technique that has unique potential for molecular cell biology. At the present resolution of 4-5 nm, large supramolecular structures can be studied in unperturbed cellular environments and, in the future, it will become possible to map molecular landscapes inside cells in a more comprehensive manner. 'Visual proteomics' aims to complement and extend mass-spectrometry-based inventories, and to provide a quantitative description of the macromolecular interactions that underlie cellular functions.

  6. Recent developments in quantitative proteomics.

    PubMed

    Becker, Christopher H; Bern, Marshall

    2011-06-17

    Proteomics is the study of proteins on a large scale, encompassing the many interests scientists and physicians have in their expression and physical properties. Proteomics continues to be a rapidly expanding field, with a wealth of reports regularly appearing on technology enhancements and scientific studies using these new tools. This review focuses primarily on the quantitative aspect of protein expression and the associated computational machinery for making large-scale identifications of proteins and their post-translational modifications. The primary emphasis is on the combination of liquid chromatography-mass spectrometry (LC-MS) methods and associated tandem mass spectrometry (LC-MS/MS). Tandem mass spectrometry, or MS/MS, involves a second analysis within the instrument after a molecular dissociative event in order to obtain structural information including but not limited to sequence information. This review further focuses primarily on the study of in vitro digested proteins known as bottom-up or shotgun proteomics. A brief discussion of recent instrumental improvements precedes a discussion on affinity enrichment and depletion of proteins, followed by a review of the major approaches (label-free and isotope-labeling) to making protein expression measurements quantitative, especially in the context of profiling large numbers of proteins. Then a discussion follows on the various computational techniques used to identify peptides and proteins from LC-MS/MS data. This review article then includes a short discussion of LC-MS approaches to three-dimensional structure determination and concludes with a section on statistics and data mining for proteomics, including comments on properly powering clinical studies and avoiding over-fitting with large data sets.

  7. Recent Developments in Quantitative Proteomics

    PubMed Central

    Becker, Christopher H.; Bern, Marshall

    2010-01-01

    Proteomics is the study of proteins on a large scale, encompassing the many interests scientists and physicians have in their expression and physical properties. Proteomics continues to be a rapidly expanding field, with a wealth of reports regularly appearing on technology enhancements and scientific studies using these new tools. This review focuses primarily on the quantitative aspect of protein expression and the associated computational machinery for making large-scale identifications of proteins and their post-translational modifications. The primary emphasis is on the combination of liquid chromatography-mass spectrometry (LC-MS) methods and associated tandem mass spectrometry (LC-MS/MS). Tandem mass spectrometry, or MS/MS, involves a second analysis within the instrument after a molecular dissociative event in order to obtain structural information including but not limited to sequence information. This review further focuses primarily on the study of in vitro digested proteins known as bottom-up or shotgun proteomics. A brief discussion of recent instrumental improvements precedes a discussion on affinity enrichment and depletion of proteins, followed by a review of the major approaches (label-free and isotope-labeling) to making protein expression measurements quantitative, especially in the context of profiling large numbers of proteins. Then a discussion follows on the various computational techniques used to identify peptides and proteins from LC-MS/MS data. This review article then includes a short discussion of LC-MS approaches to three-dimensional structure determination and concludes with a section on statistics and data mining for proteomics, including comments on properly powering clinical studies and avoiding over-fitting with large data sets. PMID:20620221

  8. Proteomics: analytical tools and techniques.

    PubMed

    MacCoss, M J; Yates, J R

    2001-09-01

    Scientists have long been interested in measuring the effects of different stimuli on protein expression and metabolism. Analytical methods are being developed for the automated separation, identification, and quantitation of all of the proteins within the cell. Soon, investigators will be able to observe the effects of an experiment on every protein (as opposed to a selected few). This review presents a discussion of recent technological advances in proteomics in addition to exploring current methodological limitations.

  9. The Cell Surface Proteome of Human Mesenchymal Stromal Cells

    PubMed Central

    Pursche, Theresia; Bornhäuser, Martin; Corbeil, Denis; Hoflack, Bernard

    2011-01-01

    Background Multipotent human mesenchymal stromal cells (hMSCs) are considered as promising biological tools for regenerative medicine. Their antibody-based isolation relies on the identification of reliable cell surface markers. Methodology/Principal Findings To obtain a comprehensive view of the cell surface proteome of bone marrow-derived hMSCs, we have developed an analytical pipeline relying on cell surface biotinylation of intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin to enrich the plasma membrane proteins and mass spectrometry for identification with extremely high confidence. Among the 888 proteins identified, we found ≈200 bona fide plasma membrane proteins including 33 cell adhesion molecules and 26 signaling receptors. In total 41 CD markers including 5 novel ones (CD97, CD112, CD239, CD276, and CD316) were identified. The CD markers are distributed homogenously within plastic-adherent hMSC populations and their expression is modulated during the process of adipogenesis or osteogenesis. Moreover, our in silico analysis revealed a significant difference between the cell surface proteome of hMSCs and that of human embryonic stem cells reported previously. Conclusions/Significance Collectively, our analytical methods not only provide a basis for further studies of mechanisms maintaining the multipotency of hMSCs within their niches and triggering their differentiation after signaling, but also a toolbox for a refined antibody-based identification of hMSC populations from different tissues and their isolation for therapeutic intervention. PMID:21637820

  10. Statistical inference from multiple iTRAQ experiments without using common reference standards.

    PubMed

    Herbrich, Shelley M; Cole, Robert N; West, Keith P; Schulze, Kerry; Yager, James D; Groopman, John D; Christian, Parul; Wu, Lee; O'Meally, Robert N; May, Damon H; McIntosh, Martin W; Ruczinski, Ingo

    2013-02-01

    Isobaric tags for relative and absolute quantitation (iTRAQ) is a prominent mass spectrometry technology for protein identification and quantification that is capable of analyzing multiple samples in a single experiment. Frequently, iTRAQ experiments are carried out using an aliquot from a pool of all samples, or "masterpool", in one of the channels as a reference sample standard to estimate protein relative abundances in the biological samples and to combine abundance estimates from multiple experiments. In this manuscript, we show that using a masterpool is counterproductive. We obtain more precise estimates of protein relative abundance by using the available biological data instead of the masterpool and do not need to occupy a channel that could otherwise be used for another biological sample. In addition, we introduce a simple statistical method to associate proteomic data from multiple iTRAQ experiments with a numeric response and show that this approach is more powerful than the conventionally employed masterpool-based approach. We illustrate our methods using data from four replicate iTRAQ experiments on aliquots of the same pool of plasma samples and from a 406-sample project designed to identify plasma proteins that covary with nutrient concentrations in chronically undernourished children from South Asia.

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

    PubMed Central

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

    2015-01-01

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

  12. Human saliva proteome: an overview

    NASA Astrophysics Data System (ADS)

    Griffin, Timothy J.

    2014-06-01

    Human saliva contains a rich mixture of biomolecules. Proteins are a major component of this mixture. Given their role as the molecular effectors within biological systems, ranging from catalysis to transport to structure, proteins have great potential as biomarkers of health and disease. The ability to collect these salivary biomarkers easily using non-invasive means makes saliva proteins even more attractive for diagnostic applications. Thousands of proteins are now to be known to be present in human saliva - discovered using proteomic technologies. Emerging technologies are now making it possible to go beyond large-scale cataloging of salivary proteins. These include approaches to catalog protein contributions from the community of microorganisms residing in the oral cavity (metaproteomics) that may reflect the health state of the human host. New mass spectrometry-based proteomics methods are also emerging, shifting the emphasis from large-scale discovery experiments to hypothesis-driven assays for profiling proteins of interest within saliva, enabling validation of their association with specific health conditions. This paper provides a brief overview of efforts to catalog the proteome of human saliva. Recent developments making possible characterization of the metaproteome of human saliva will be discussed, and technologies driving new mass spectrometry-based assays for targeted analysis of proteins within complex samples, such as saliva.

  13. Protein Neighbors and Proximity Proteomics*

    PubMed Central

    Rees, Johanna S.; Li, Xue-Wen; Perrett, Sarah; Lilley, Kathryn S.; Jackson, Antony P.

    2015-01-01

    Within cells, proteins can co-assemble into functionally integrated and spatially restricted multicomponent complexes. Often, the affinities between individual proteins are relatively weak, and proteins within such clusters may interact only indirectly with many of their other protein neighbors. This makes proteomic characterization difficult using methods such as immunoprecipitation or cross-linking. Recently, several groups have described the use of enzyme-catalyzed proximity labeling reagents that covalently tag the neighbors of a targeted protein with a small molecule such as fluorescein or biotin. The modified proteins can then be isolated by standard pulldown methods and identified by mass spectrometry. Here we will describe the techniques as well as their similarities and differences. We discuss their applications both to study protein assemblies and to provide a new way for characterizing organelle proteomes. We stress the importance of proteomic quantitation and independent target validation in such experiments. Furthermore, we suggest that there are biophysical and cell-biological principles that dictate the appropriateness of enzyme-catalyzed proximity labeling methods to address particular biological questions of interest. PMID:26355100

  14. CMPD: cancer mutant proteome database.

    PubMed

    Huang, Po-Jung; Lee, Chi-Ching; Tan, Bertrand Chin-Ming; Yeh, Yuan-Ming; Julie Chu, Lichieh; Chen, Ting-Wen; Chang, Kai-Ping; Lee, Cheng-Yang; Gan, Ruei-Chi; Liu, Hsuan; Tang, Petrus

    2015-01-01

    Whole-exome sequencing, which centres on the protein coding regions of disease/cancer associated genes, represents the most cost-effective method to-date for deciphering the association between genetic alterations and diseases. Large-scale whole exome/genome sequencing projects have been launched by various institutions, such as NCI, Broad Institute and TCGA, to provide a comprehensive catalogue of coding variants in diverse tissue samples and cell lines. Further functional and clinical interrogation of these sequence variations must rely on extensive cross-platforms integration of sequencing information and a proteome database that explicitly and comprehensively archives the corresponding mutated peptide sequences. While such data resource is a critical for the mass spectrometry-based proteomic analysis of exomic variants, no database is currently available for the collection of mutant protein sequences that correspond to recent large-scale genomic data. To address this issue and serve as bridge to integrate genomic and proteomics datasets, CMPD (http://cgbc.cgu.edu.tw/cmpd) collected over 2 millions genetic alterations, which not only facilitates the confirmation and examination of potential cancer biomarkers but also provides an invaluable resource for translational medicine research and opportunities to identify mutated proteins encoded by mutated genes.

  15. CMPD: cancer mutant proteome database

    PubMed Central

    Huang, Po-Jung; Lee, Chi-Ching; Tan, Bertrand Chin-Ming; Yeh, Yuan-Ming; Julie Chu, Lichieh; Chen, Ting-Wen; Chang, Kai-Ping; Lee, Cheng-Yang; Gan, Ruei-Chi; Liu, Hsuan; Tang, Petrus

    2015-01-01

    Whole-exome sequencing, which centres on the protein coding regions of disease/cancer associated genes, represents the most cost-effective method to-date for deciphering the association between genetic alterations and diseases. Large-scale whole exome/genome sequencing projects have been launched by various institutions, such as NCI, Broad Institute and TCGA, to provide a comprehensive catalogue of coding variants in diverse tissue samples and cell lines. Further functional and clinical interrogation of these sequence variations must rely on extensive cross-platforms integration of sequencing information and a proteome database that explicitly and comprehensively archives the corresponding mutated peptide sequences. While such data resource is a critical for the mass spectrometry-based proteomic analysis of exomic variants, no database is currently available for the collection of mutant protein sequences that correspond to recent large-scale genomic data. To address this issue and serve as bridge to integrate genomic and proteomics datasets, CMPD (http://cgbc.cgu.edu.tw/cmpd) collected over 2 millions genetic alterations, which not only facilitates the confirmation and examination of potential cancer biomarkers but also provides an invaluable resource for translational medicine research and opportunities to identify mutated proteins encoded by mutated genes. PMID:25398898

  16. Forensic Proteomics of Poxvirus Production

    SciTech Connect

    Wunschel, David S.; Tulman, Edan; Engelmann, Heather E.; Clowers, Brian H.; Geary, Steven J.; Robinson, Aaron C.; Liao, Xiaofen

    2013-08-27

    The field of microbial forensics has recently sought to develop methods to discern biological signatures to indicate production methods for biological agents. Viral agents have received less attention to date. Their obligate propagation in living cells makes purification from cellular material a challenge. This leads to potential carryover of protein-rich signature of their production system. Here we have explored a proteomic analysis of Vaccinia virus as a model poxvirus system in which to compare samples of virus propagated in different cell lines and subjected to different purification schemes. The proteomic data sets indicated viral, host cell and culture medium proteins, and several layers of data analysis were applied to build confidence in the peptide identification and capture information on the taxonomic utility of each. The analysis showed clear shifts in protein profiles with virus purification, with successive gradient purification steps showing different levels of viral protein enrichment. Peptides from cellular proteins, including those present in purified virus preparations, provided signatures which enabled discrimination of cell line substrates, including distinguishing between cells derived from different primate species. The ability to discern multiple aspects of viral production demonstrates the potential value of proteomic analysis as tool for microbial forensics.

  17. Applications of Proteomic Technologies to Toxicology

    EPA Science Inventory

    Proteomics is the large-scale study of gene expression at the protein level. This cutting edge technology has been extensively applied to toxicology research recently. The up-to-date development of proteomics has presented the toxicology community with an unprecedented opportunit...

  18. Global Proteome Analysis of Leptospira interrogans

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  19. Proteomics: Protein Identification Using Online Databases

    ERIC Educational Resources Information Center

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

    2012-01-01

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

  20. Endosperm and Amyloplast Proteomes of Wheat Grain

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Advances in proteomics and genomics have improved our understanding of the gluten proteins, a complex and functionally important protein group. Proteomic approaches also have been used to identify other proteins that may play roles in wheat flour functionality, to assign genes for gluten proteins to...

  1. Centennial Paper: Proteomics in animal science

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteomics holds significant promise as a method for advancing animal science research. The use of this technology in animal science is still in its infancy. The ability of proteomics to simultaneously identify and quantify potentially thousands of proteins is unparalleled. In this review, we wil...

  2. The promise of proteomics in animal science

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteomics hold significant promise as a method for advancing animal science research. The use of this technology in animal science is still in its infancy. The ability of proteomics to simultaneously identify and quantify potentially thousands of proteins is unparalleled. In this review, we will...

  3. Structural characterization of the human proteome.

    PubMed

    Müller, Arne; MacCallum, Robert M; Sternberg, Michael J E

    2002-11-01

    This paper reports an analysis of the encoded proteins (the proteome) of the genomes of human, fly, worm, yeast, and representatives of bacteria and archaea in terms of the three-dimensional structures of their globular domains together with a general sequence-based study. We show that 39% of the human proteome can be assigned to known structures. We estimate that for 77% of the proteome, there is some functional annotation, but only 26% of the proteome can be assigned to standard sequence motifs that characterize function. Of the human protein sequences, 13% are transmembrane proteins, but only 3% of the residues in the proteome form membrane-spanning regions. There are substantial differences in the composition of globular domains of transmembrane proteins between the proteomes we have analyzed. Commonly occurring structural superfamilies are identified within the proteome. The frequencies of these superfamilies enable us to estimate that 98% of the human proteome evolved by domain duplication, with four of the 10 most duplicated superfamilies specific for multicellular organisms. The zinc-finger superfamily is massively duplicated in human compared to fly and worm, and occurrence of domains in repeats is more common in metazoa than in single cellular organisms. Structural superfamilies over- and underrepresented in human disease genes have been identified. Data and results can be downloaded and analyzed via web-based applications at http://www.sbg.bio.ic.ac.uk.

  4. Proteotyping: Proteomic characterization, classification and identification of microorganisms--A prospectus.

    PubMed

    Karlsson, Roger; Gonzales-Siles, Lucia; Boulund, Fredrik; Svensson-Stadler, Liselott; Skovbjerg, Susann; Karlsson, Anders; Davidson, Max; Hulth, Stefan; Kristiansson, Erik; Moore, Edward R B

    2015-06-01

    Modern microbial systematics requires a range of methodologies for the comprehensive characterization, classification and identification of microorganisms. While whole-genome sequences provide the ultimate reference for defining microbial phylogeny and taxonomy, selected biomarker-based strategies continue to provide the means for the bulk of microbial systematic studies. Proteomics, the study of the expression of genes, as well as the structure and function of the resulting proteins, offers indirect measures of genome sequence data. Recent developments in applications of proteomics for analyzing microorganisms have paralleled the growing microbial genome sequence database, as well as the evolution of mass spectrometry (MS) instrumentation and bioinformatics. MALDI-TOF MS, which generates proteomic mass patterns for 'fingerprint'-based characterizations, has provided a marked breakthrough for microbial identification. However, MALDI-TOF MS is limited in the number of targets that can be detected for strain characterization. Advanced methods of tandem mass spectrometry, in which proteins and peptides generated from proteins, are characterized and identified, using LC-MS/MS, provide the ability to detect hundreds or thousands of expressed microbial strain markers for high-resolution characterizations and identifications. Model studies demonstrate the application of proteomics-based analyses for bacterial species- and strain-level detection and identification and for characterization of environmentally relevant, metabolically diverse bacteria. Proteomics-based approaches represent an emerging complement to traditional methods of characterizing microorganisms, enabling the elucidation of the expressed biomarkers of genome sequence information, which can be applied to 'proteotyping' applications of microorganisms at all taxonomic levels.

  5. Characterization of the Human Pancreatic Islet Proteome by Two-Dimensional LC/MS/MS

    SciTech Connect

    Metz, Thomas O.; Jacobs, Jon M.; Gritsenko, Marina A.; Fontes, Ghislaine; Qian, Weijun; Camp, David G.; Poitout, Vincent J.; Smith, Richard D.

    2006-12-01

    Research to elucidate the pathogenesis of type 1 diabetes mellitus has traditionally focused on the genetic and immunological factors associated with the disease, and, until recently, has not considered the target cell. While there have been reports detailing proteomic analyses of established islet cell lines or isolated rodent islets, the information gained is not always easily extrapolated to humans. Therefore, extensive characterization of the human islet proteome could result in better understanding of islet biology and lead to more effective treatment strategies. We have applied a two-dimensional LC-MS/MS-based analysis to the characterization of the human islet proteome, resulting in the detection of 29,021 unique peptides corresponding to 4,925 proteins. As expected, major islet hormones (insulin, glucagon, somatostatin), beta-cell enriched secretory products (IAPP), ion channels (K-ATP channel), and transcription factors (PDX-1, Nkx 6.1, HNF-1 beta) were detected. In addition, significant proteome coverage of metabolic enzymes and cellular pathways was obtained, including the insulin signaling cascade and the MAP kinase, NF-κβ, and JAK/STAT pathways. This work represents the most extensive characterization of the human islet proteome to date and provides a peptide reference library that may be utilized in future studies of islet biology and type 1 diabetes.

  6. Proteome dataset of pre-ovulatory follicular fluids from less fertile dairy cows.

    PubMed

    Zachut, Maya; Sood, Pankaj; Livshitz, Lilya; Kra, Gitit; Levin, Yishai; Moallem, Uzi

    2016-06-01

    This article contains raw and processed data related to research published in Zachut et al. (2016) [1]. Proteomics data from preovulatory follicles in cows was obtained by liquid chromatography-mass spectrometry following protein extraction. Differential expression between controls and less fertile cows (LFC) was quantified using MS1 intensity based label-free. The only previous proteomic analysis of bovine FF detected merely 40 proteins in follicular cysts obtained from the slaughterhouse (Maniwa et al., 2005) [2], and the abundance of proteins in the bovine preovulatory FF remains unknown. Therefore, the objectives were to establish the first dataset of FF proteome in preovulatory follicles of cows, and to examine differentially expressed proteins in FF obtained in-vivo from preovulatory follicles of less fertile cows (also termed "repeat breeder") and control (CTL) cows. The proteome of FF from 10 preovulatory follicles that were aspirated in vivo (estradiol/progesterone>1) was analyzed. This novel dataset contains 219 identified and quantified proteins in FF, consisting mainly of binding proteins, proteases, receptor ligands, enzymes and transporters. In addition, differential abundance of 8 proteins relevant to follicular function was found in LFC compared to CTL; these findings are discussed in our recent research article Zachut et al. (2016) [1]. The present dataset of bovine FF proteome can be used as a reference for any study involving disorders of follicular development in dairy cows or in comparative studies between species. PMID:27182550

  7. Proteome dataset of pre-ovulatory follicular fluids from less fertile dairy cows.

    PubMed

    Zachut, Maya; Sood, Pankaj; Livshitz, Lilya; Kra, Gitit; Levin, Yishai; Moallem, Uzi

    2016-06-01

    This article contains raw and processed data related to research published in Zachut et al. (2016) [1]. Proteomics data from preovulatory follicles in cows was obtained by liquid chromatography-mass spectrometry following protein extraction. Differential expression between controls and less fertile cows (LFC) was quantified using MS1 intensity based label-free. The only previous proteomic analysis of bovine FF detected merely 40 proteins in follicular cysts obtained from the slaughterhouse (Maniwa et al., 2005) [2], and the abundance of proteins in the bovine preovulatory FF remains unknown. Therefore, the objectives were to establish the first dataset of FF proteome in preovulatory follicles of cows, and to examine differentially expressed proteins in FF obtained in-vivo from preovulatory follicles of less fertile cows (also termed "repeat breeder") and control (CTL) cows. The proteome of FF from 10 preovulatory follicles that were aspirated in vivo (estradiol/progesterone>1) was analyzed. This novel dataset contains 219 identified and quantified proteins in FF, consisting mainly of binding proteins, proteases, receptor ligands, enzymes and transporters. In addition, differential abundance of 8 proteins relevant to follicular function was found in LFC compared to CTL; these findings are discussed in our recent research article Zachut et al. (2016) [1]. The present dataset of bovine FF proteome can be used as a reference for any study involving disorders of follicular development in dairy cows or in comparative studies between species.

  8. Development of proteome-wide binding reagents for research and diagnostics.

    PubMed

    Taussig, Michael J; Schmidt, Ronny; Cook, Elizabeth A; Stoevesandt, Oda

    2013-12-01

    Alongside MS, antibodies and other specific protein-binding molecules have a special place in proteomics as affinity reagents in a toolbox of applications for determining protein location, quantitative distribution and function (affinity proteomics). The realisation that the range of research antibodies available, while apparently vast is nevertheless still very incomplete and frequently of uncertain quality, has stimulated projects with an objective of raising comprehensive, proteome-wide sets of protein binders. With progress in automation and throughput, a remarkable number of recent publications refer to the practical possibility of selecting binders to every protein encoded in the genome. Here we review the requirements of a pipeline of production of protein binders for the human proteome, including target prioritisation, antigen design, 'next generation' methods, databases and the approaches taken by ongoing projects in Europe and the USA. While the task of generating affinity reagents for all human proteins is complex and demanding, the benefits of well-characterised and quality-controlled pan-proteome binder resources for biomedical research, industry and life sciences in general would be enormous and justify the effort. Given the technical, personnel and financial resources needed to fulfil this aim, expansion of current efforts may best be addressed through large-scale international collaboration.

  9. Reference Frames and Relativity.

    ERIC Educational Resources Information Center

    Swartz, Clifford

    1989-01-01

    Stresses the importance of a reference frame in mechanics. Shows the Galilean transformation in terms of relativity theory. Discusses accelerated reference frames and noninertial reference frames. Provides examples of reference frames with diagrams. (YP)

  10. Proteome Analysis of the Penicillin Producer Penicillium chrysogenum

    PubMed Central

    Jami, Mohammad-Saeid; Barreiro, Carlos; García-Estrada, Carlos; Martín, Juan-Francisco

    2010-01-01

    Proteomics is a powerful tool to understand the molecular mechanisms causing the production of high penicillin titers by industrial strains of the filamentous fungus Penicillium chrysogenum as the result of strain improvement programs. Penicillin biosynthesis is an excellent model system for many other bioactive microbial metabolites. The recent publication of the P. chrysogenum genome has established the basis to understand the molecular processes underlying penicillin overproduction. We report here the proteome reference map of P. chrysogenum Wisconsin 54-1255 (the genome project reference strain) together with an in-depth study of the changes produced in three different strains of this filamentous fungus during industrial strain improvement. Two-dimensional gel electrophoresis, peptide mass fingerprinting, and tandem mass spectrometry were used for protein identification. Around 1000 spots were visualized by “blue silver” colloidal Coomassie staining in a non-linear pI range from 3 to 10 with high resolution, which allowed the identification of 950 proteins (549 different proteins and isoforms). Comparison among the cytosolic proteomes of the wild-type NRRL 1951, Wisconsin 54-1255 (an improved, moderate penicillin producer), and AS-P-78 (a penicillin high producer) strains indicated that global metabolic reorganizations occurred during the strain improvement program. The main changes observed in the high producer strains were increases of cysteine biosynthesis (a penicillin precursor), enzymes of the pentose phosphate pathway, and stress response proteins together with a reduction in virulence and in the biosynthesis of other secondary metabolites different from penicillin (pigments and isoflavonoids). In the wild-type strain, we identified enzymes to utilize cellulose, sorbitol, and other carbon sources that have been lost in the high penicillin producer strains. Changes in the levels of a few specific proteins correlated well with the improved penicillin

  11. Utilization of proteomics in experimental field conditions--A case study of poplars growing on grassland affected by long-term starch wastewater irrigation.

    PubMed

    Szuba, Agnieszka; Lorenc-Plucińska, Gabriela

    2015-08-01

    The presented study verified the possibility of using proteomics as a tool for investigating poplars growing on obviously separate plots. The examination covered poplars planted on grassland irrigated for 40 years with potato industry wastewater and in a plot appropriate for poplar planting, spaced at a distance of 67 km from each other (hereinafter referred to as forest). The work aimed to compare the obtained proteomic results with data on biometric and biochemical parameters and mineral composition as well as to assess, at a molecular level, the usefulness of grasslands for planting. Proteome analysis showed that most of the stress-related proteins detected were less abundant on the irrigated grassland, confirming the viability of its revegetation with poplars. Proteomic data corresponded well with the other results, highlighting the probable reason for the proteome changes; i.e. deficiency of phosphate ions detected in the forest area. Moreover, proteome analysis revealed biotic stress symptoms in plants growing on the grassland, which were also well explained by other data but would not have been detected without performing the proteomic analysis. Therefore, environmental plant proteomics is a useful and valuable tool during field studies, even when samples are taken from plots some distance apart.

  12. Accurate Mass Measurements in Proteomics

    SciTech Connect

    Liu, Tao; Belov, Mikhail E.; Jaitly, Navdeep; Qian, Weijun; Smith, Richard D.

    2007-08-01

    To understand different aspects of life at the molecular level, one would think that ideally all components of specific processes should be individually isolated and studied in details. Reductionist approaches, i.e., studying one biological event at a one-gene or one-protein-at-a-time basis, indeed have made significant contributions to our understanding of many basic facts of biology. However, these individual “building blocks” can not be visualized as a comprehensive “model” of the life of cells, tissues, and organisms, without using more integrative approaches.1,2 For example, the emerging field of “systems biology” aims to quantify all of the components of a biological system to assess their interactions and to integrate diverse types of information obtainable from this system into models that could explain and predict behaviors.3-6 Recent breakthroughs in genomics, proteomics, and bioinformatics are making this daunting task a reality.7-14 Proteomics, the systematic study of the entire complement of proteins expressed by an organism, tissue, or cell under a specific set of conditions at a specific time (i.e., the proteome), has become an essential enabling component of systems biology. While the genome of an organism may be considered static over short timescales, the expression of that genome as the actual gene products (i.e., mRNAs and proteins) is a dynamic event that is constantly changing due to the influence of environmental and physiological conditions. Exclusive monitoring of the transcriptomes can be carried out using high-throughput cDNA microarray analysis,15-17 however the measured mRNA levels do not necessarily correlate strongly with the corresponding abundances of proteins,18-20 The actual amount of functional proteins can be altered significantly and become independent of mRNA levels as a result of post-translational modifications (PTMs),21 alternative splicing,22,23 and protein turnover.24,25 Moreover, the functions of expressed

  13. Toxic metal proteomics: reaction of the mammalian zinc proteome with Cd²⁺.

    PubMed

    Namdarghanbari, Mohammad Ali; Bertling, Joseph; Krezoski, Susan; Petering, David H

    2014-07-01

    The hypothesis was tested that Cd(2+) undergoes measureable reaction with the Zn-proteome through metal ion exchange chemistry. The Zn-proteome of pig kidney LLC-PK1 cells is relatively inert to reaction with competing ligands, including Zinquin acid, EDTA, and apo-metallothionein. Upon reaction of Cd(2+) with the Zn-proteome, Cd(2+) associates with the proteome and near stoichiometric amounts of Zn(2+) become reactive with these chelating agents. The results strongly support the hypothesis that Cd(2+) displaces Zn(2+) from native proteomic binding sites resulting in the formation of a Cd-proteome. Mobilized Zn(2+) becomes adventitiously bound to proteome and available for reaction with added metal binding ligands. Cd-proteome and Zn-metallothionein readily exchange metal ions, raising the possibility that this reaction restores functionality to Cd-proteins. In a parallel experiment, cells were exposed to Cd(2+) and pyrithione briefly to generate substantial proteome-bound Cd(2+). Upon transition to a Cd(2+) free medium, the cells generated new metallothionein protein over time that bound most of the proteomic Cd(2+) as well as additional Zn(2+). PMID:24529759

  14. The International Proteomics Tutorial Programme (IPTP): a teaching tool box for th