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Sample records for affinity purification-mass spectrometry

  1. Solid support resins and affinity purification mass spectrometry.

    PubMed

    Havis, Spencer; Moree, Wilna J; Mali, Sujina; Bark, Steven J

    2017-02-28

    Co-affinity purification-mass spectrometry (CoAP-MS) is a primary technology for elucidating the protein-protein interactions that form the basis of all biological processes. A critical component of CoAP-MS is the affinity purification (AP) of the bait protein, usually by immobilization of an antibody to a solid-phase resin. This Minireview discusses common resins, reagents, tagging methods, and their consideration for successful AP of tagged proteins. We discuss our experiences with different solid supports, their impact in AP experiments, and propose areas where chemistry can advance this important technology.

  2. Observations on different resin strategies for affinity purification mass spectrometry of a tagged protein.

    PubMed

    Mali, Sujina; Moree, Wilna J; Mitchell, Morgan; Widger, William; Bark, Steven J

    2016-12-15

    Co-affinity purification mass spectrometry (CoAP-MS) is a highly effective method for identifying protein complexes from a biological sample and inferring important interactions, but the impact of the solid support is usually not considered in design of such experiments. Affinity purification (AP) experiments typically utilize a bait protein expressing a peptide tag such as FLAG, c-Myc, HA or V5 and high affinity antibodies to these peptide sequences to facilitate isolation of a bait protein to co-purify interacting proteins. We observed significant variability for isolation of tagged bait proteins between Protein A/G Agarose, Protein G Dynabeads, and AminoLink resins. While previous research identified the importance of tag sequence and their location, crosslinking procedures, reagents, dilution, and detergent concentrations, the effect of the resin itself has not been considered. Our data suggest the type of solid support is important and, under the conditions of our experiments, AminoLink resin provided a more robust solid-support platform for AP-MS.

  3. The CRAPome: a Contaminant Repository for Affinity Purification Mass Spectrometry Data

    PubMed Central

    Mellacheruvu, Dattatreya; Wright, Zachary; Couzens, Amber L.; Lambert, Jean-Philippe; St-Denis, Nicole; Li, Tuo; Miteva, Yana V.; Hauri, Simon; Sardiu, Mihaela E.; Low, Teck Yew; Halim, Vincentius A.; Bagshaw, Richard D.; Hubner, Nina C.; al-Hakim, Abdallah; Bouchard, Annie; Faubert, Denis; Fermin, Damian; Dunham, Wade H.; Goudreault, Marilyn; Lin, Zhen-Yuan; Badillo, Beatriz Gonzalez; Pawson, Tony; Durocher, Daniel; Coulombe, Benoit; Aebersold, Ruedi; Superti-Furga, Giulio; Colinge, Jacques; Heck, Albert J. R.; Choi, Hyungwon; Gstaiger, Matthias; Mohammed, Shabaz; Cristea, Ileana M.; Bennett, Keiryn L.; Washburn, Mike P.; Raught, Brian; Ewing, Rob M.; Gingras, Anne-Claude; Nesvizhskii, Alexey I.

    2013-01-01

    Affinity purification coupled with mass spectrometry (AP-MS) is now a widely used approach for the identification of protein-protein interactions. However, for any given protein of interest, determining which of the identified polypeptides represent bona fide interactors versus those that are background contaminants (e.g. proteins that interact with the solid-phase support, affinity reagent or epitope tag) is a challenging task. While the standard approach is to identify nonspecific interactions using one or more negative controls, most small-scale AP-MS studies do not capture a complete, accurate background protein set. Fortunately, negative controls are largely bait-independent. Hence, aggregating negative controls from multiple AP-MS studies can increase coverage and improve the characterization of background associated with a given experimental protocol. Here we present the Contaminant Repository for Affinity Purification (the CRAPome) and describe the use of this resource to score protein-protein interactions. The repository (currently available for Homo sapiens and Saccharomyces cerevisiae) and computational tools are freely available online at www.crapome.org. PMID:23921808

  4. The CRAPome: a contaminant repository for affinity purification-mass spectrometry data.

    PubMed

    Mellacheruvu, Dattatreya; Wright, Zachary; Couzens, Amber L; Lambert, Jean-Philippe; St-Denis, Nicole A; Li, Tuo; Miteva, Yana V; Hauri, Simon; Sardiu, Mihaela E; Low, Teck Yew; Halim, Vincentius A; Bagshaw, Richard D; Hubner, Nina C; Al-Hakim, Abdallah; Bouchard, Annie; Faubert, Denis; Fermin, Damian; Dunham, Wade H; Goudreault, Marilyn; Lin, Zhen-Yuan; Badillo, Beatriz Gonzalez; Pawson, Tony; Durocher, Daniel; Coulombe, Benoit; Aebersold, Ruedi; Superti-Furga, Giulio; Colinge, Jacques; Heck, Albert J R; Choi, Hyungwon; Gstaiger, Matthias; Mohammed, Shabaz; Cristea, Ileana M; Bennett, Keiryn L; Washburn, Mike P; Raught, Brian; Ewing, Rob M; Gingras, Anne-Claude; Nesvizhskii, Alexey I

    2013-08-01

    Affinity purification coupled with mass spectrometry (AP-MS) is a widely used approach for the identification of protein-protein interactions. However, for any given protein of interest, determining which of the identified polypeptides represent bona fide interactors versus those that are background contaminants (for example, proteins that interact with the solid-phase support, affinity reagent or epitope tag) is a challenging task. The standard approach is to identify nonspecific interactions using one or more negative-control purifications, but many small-scale AP-MS studies do not capture a complete, accurate background protein set when available controls are limited. Fortunately, negative controls are largely bait independent. Hence, aggregating negative controls from multiple AP-MS studies can increase coverage and improve the characterization of background associated with a given experimental protocol. Here we present the contaminant repository for affinity purification (the CRAPome) and describe its use for scoring protein-protein interactions. The repository (currently available for Homo sapiens and Saccharomyces cerevisiae) and computational tools are freely accessible at http://www.crapome.org/.

  5. Intelligent Mixing of Proteomes for Elimination of False Positives in Affinity Purification-Mass Spectrometry.

    PubMed

    Eyckerman, Sven; Impens, Francis; Van Quickelberghe, Emmy; Samyn, Noortje; Vandemoortele, Giel; De Sutter, Delphine; Tavernier, Jan; Gevaert, Kris

    2016-10-07

    Protein complexes are essential in all organizational and functional aspects of the cell. Different strategies currently exist for analyzing such protein complexes by mass spectrometry, including affinity purification (AP-MS) and proximal labeling-based strategies. However, the high sensitivity of current mass spectrometers typically results in extensive protein lists mainly consisting of nonspecifically copurified proteins. Finding the true positive interactors in these lists remains highly challenging. Here, we report a powerful design based on differential labeling with stable isotopes combined with nonequal mixing of control and experimental samples to discover bona fide interaction partners in AP-MS experiments. We apply this intelligent mixing of proteomes (iMixPro) concept to overexpression experiments for RAF1, RNF41, and TANK and also to engineered cell lines expressing epitope-tagged endogenous PTPN14, JIP3, and IQGAP1. For all baits, we confirmed known interactions and found a number of novel interactions. The results for RNF41 and TANK were compared to a classical affinity purification experiment, which demonstrated the efficiency and specificity of the iMixPro approach.

  6. Using ProHits to store, annotate, and analyze affinity purification-mass spectrometry (AP-MS) data.

    PubMed

    Liu, Guomin; Zhang, Jianping; Choi, Hyungwon; Lambert, Jean-Philippe; Srikumar, Tharan; Larsen, Brett; Nesvizhskii, Alexey I; Raught, Brian; Tyers, Mike; Gingras, Anne-Claude

    2012-09-01

    Affinity purification coupled with mass spectrometry (AP-MS) is a robust technique used to identify protein-protein interactions. With recent improvements in sample preparation, and dramatic advances in MS instrumentation speed and sensitivity, this technique is becoming more widely used throughout the scientific community. To meet the needs of research groups both large and small, we have developed software solutions for tracking, scoring and analyzing AP-MS data. Here, we provide details for the installation and utilization of ProHits, a Laboratory Information Management System designed specifically for AP-MS interaction proteomics. This protocol explains: (i) how to install the complete ProHits system, including modules for the management of mass spectrometry files and the analysis of interaction data, and (ii) alternative options for the use of pre-existing search results in simpler versions of ProHits, including a virtual machine implementation of our ProHits Lite software. We also describe how to use the main features of the software to analyze AP-MS data.

  7. Computational and informatics strategies for identification of specific protein interaction partners in affinity purification mass spectrometry experiments

    PubMed Central

    Nesvizhskii, Alexey I.

    2013-01-01

    Analysis of protein interaction networks and protein complexes using affinity purification and mass spectrometry (AP/MS) is among most commonly used and successful applications of proteomics technologies. One of the foremost challenges of AP/MS data is a large number of false positive protein interactions present in unfiltered datasets. Here we review computational and informatics strategies for detecting specific protein interaction partners in AP/MS experiments, with a focus on incomplete (as opposite to genome-wide) interactome mapping studies. These strategies range from standard statistical approaches, to empirical scoring schemes optimized for a particular type of data, to advanced computational frameworks. The common denominator among these methods is the use of label-free quantitative information such as spectral counts or integrated peptide intensities that can be extracted from AP/MS data. We also discuss related issues such as combining multiple biological or technical replicates, and dealing with data generated using different tagging strategies. Computational approaches for benchmarking of scoring methods are discussed, and the need for generation of reference AP/MS datasets is highlighted. Finally, we discuss the possibility of more extended modeling of experimental AP/MS data, including integration with external information such as protein interaction predictions based on functional genomics data. PMID:22611043

  8. PIPINO: A Software Package to Facilitate the Identification of Protein-Protein Interactions from Affinity Purification Mass Spectrometry Data

    PubMed Central

    Schildbach, Stefan; Blumert, Conny; Horn, Friedemann; von Bergen, Martin; Labudde, Dirk

    2016-01-01

    The functionality of most proteins is regulated by protein-protein interactions. Hence, the comprehensive characterization of the interactome is the next milestone on the path to understand the biochemistry of the cell. A powerful method to detect protein-protein interactions is a combination of coimmunoprecipitation or affinity purification with quantitative mass spectrometry. Nevertheless, both methods tend to precipitate a high number of background proteins due to nonspecific interactions. To address this challenge the software Protein-Protein-Interaction-Optimizer (PIPINO) was developed to perform an automated data analysis, to facilitate the selection of bona fide binding partners, and to compare the dynamic of interaction networks. In this study we investigated the STAT1 interaction network and its activation dependent dynamics. Stable isotope labeling by amino acids in cell culture (SILAC) was applied to analyze the STAT1 interactome after streptavidin pull-down of biotagged STAT1 from human embryonic kidney 293T cells with and without activation. Starting from more than 2,000 captured proteins 30 potential STAT1 interaction partners were extracted. Interestingly, more than 50% of these were already reported or predicted to bind STAT1. Furthermore, 16 proteins were found to affect the binding behavior depending on STAT1 phosphorylation such as STAT3 or the importin subunits alpha 1 and alpha 6. PMID:26966684

  9. SAINT-MS1: protein-protein interaction scoring using label-free intensity data in affinity purification-mass spectrometry experiments.

    PubMed

    Choi, Hyungwon; Glatter, Timo; Gstaiger, Mathias; Nesvizhskii, Alexey I

    2012-04-06

    We present a statistical method SAINT-MS1 for scoring protein-protein interactions based on the label-free MS1 intensity data from affinity purification-mass spectrometry (AP-MS) experiments. The method is an extension of Significance Analysis of INTeractome (SAINT), a model-based method previously developed for spectral count data. We reformulated the statistical model for log-transformed intensity data, including adequate treatment of missing observations, that is, interactions identified in some but not all replicate purifications. We demonstrate the performance of SAINT-MS1 using two recently published data sets: a small LTQ-Orbitrap data set with three replicate purifications of single human bait protein and control purifications and a larger drosophila data set targeting insulin receptor/target of rapamycin signaling pathway generated using an LTQ-FT instrument. Using the drosophila data set, we also compare and discuss the performance of SAINT analysis based on spectral count and MS1 intensity data in terms of the recovery of orthologous and literature-curated interactions. Given rapid advances in high mass accuracy instrumentation and intensity-based label-free quantification software, we expect that SAINT-MS1 will become a useful tool allowing improved detection of protein interactions in label-free AP-MS data, especially in the low abundance range.

  10. Interactome analysis of AMP-activated protein kinase (AMPK)-α1 and -β1 in INS-1 pancreatic beta-cells by affinity purification-mass spectrometry.

    PubMed

    Moon, Sungyoon; Han, Dohyun; Kim, Yikwon; Jin, Jonghwa; Ho, Won-Kyung; Kim, Youngsoo

    2014-03-14

    The heterotrimeric enzyme AMP-activated protein kinase (AMPK) is a major metabolic factor that regulates the homeostasis of cellular energy. In particular, AMPK mediates the insulin resistance that is associated with type 2 diabetes. Generally, cellular processes require tight regulation of protein kinases, which is effected through their formation of complex with other proteins and substrates. Despite their critical function in regulation and pathogenesis, there are limited data on the interaction of protein kinases. To identify proteins that interact with AMPK, we performed large-scale affinity purification (AP)-mass spectrometry (MS) of the AMPK-α1 and -β1 subunits. Through a comprehensive analysis, using a combination of immunoprecipitaion and ion trap mass spectrometry, we identified 381 unique proteins in the AMPKα/β interactomes: 325 partners of AMPK-α1 and 243 for AMPK-β1. Further, we identified 196 novel protein-protein interactions with AMPK-α1 and AMPK-β1. Notably, in our bioinformatics analysis, the novel interaction partners mediated functions that are related to the regulation of actin organization. Specifically, several such proteins were linked to pancreatic beta-cell functions, including glucose-stimulated insulin secretion, beta-cell development, beta-cell differentiation, and cell-cell communication.

  11. Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-coupled Affinity Purification/Mass Spectrometry Analysis Revealed a Novel Role of Neurofibromin in mTOR Signaling.

    PubMed

    Li, Xu; Gao, Min; Choi, Jong Min; Kim, Beom-Jun; Zhou, Mao-Tian; Chen, Zhen; Jain, Antrix N; Jung, Sung Yun; Yuan, Jingsong; Wang, Wenqi; Wang, Yi; Chen, Junjie

    2017-04-01

    Neurofibromin (NF1) is a well known tumor suppressor that is commonly mutated in cancer patients. It physically interacts with RAS and negatively regulates RAS GTPase activity. Despite the importance of NF1 in cancer, a high quality endogenous NF1 interactome has yet to be established. In this study, we combined clustered, regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated gene knock-out technology with affinity purification using antibodies against endogenous proteins, followed by mass spectrometry analysis, to sensitively and accurately detect NF1 protein-protein interactions in unaltered in vivo settings. Using this system, we analyzed endogenous NF1-associated protein complexes and identified 49 high-confidence candidate interaction proteins, including RAS and other functionally relevant proteins. Through functional validation, we found that NF1 negatively regulates mechanistic target of rapamycin signaling (mTOR) in a LAMTOR1-dependent manner. In addition, the cell growth and survival of NF1-deficient cells have become dependent on hyperactivation of the mTOR pathway, and the tumorigenic properties of these cells have become dependent on LAMTOR1. Taken together, our findings may provide novel insights into therapeutic approaches targeting NF1-deficient tumors.

  12. An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client.

    PubMed

    Bigenzahn, Johannes W; Fauster, Astrid; Rebsamen, Manuele; Kandasamy, Richard K; Scorzoni, Stefania; Vladimer, Gregory I; Müller, André C; Gstaiger, Matthias; Zuber, Johannes; Bennett, Keiryn L; Superti-Furga, Giulio

    2016-03-01

    Tandem affinity purification-mass spectrometry (TAP-MS) is a popular strategy for the identification of protein-protein interactions, characterization of protein complexes, and entire networks. Its employment in cellular settings best fitting the relevant physiology is limited by convenient expression vector systems. We developed an easy-to-handle, inducible, dually selectable retroviral expression vector allowing dose- and time-dependent control of bait proteins bearing the efficient streptavidin-hemagglutinin (SH)-tag at their N- or C termini. Concomitant expression of a reporter fluorophore allows to monitor bait-expressing cells by flow cytometry or microscopy and enables high-throughput phenotypic assays. We used the system to successfully characterize the interactome of the neuroblastoma RAS viral oncogene homolog (NRAS) Gly12Asp (G12D) mutant and exploited the advantage of reporter fluorophore expression by tracking cytokine-independent cell growth using flow cytometry. Moreover, we tested the feasibility of studying cytotoxicity-mediating proteins with the vector system on the cell death-inducing mixed lineage kinase domain-like protein (MLKL) Ser358Asp (S358D) mutant. Interaction proteomics analysis of MLKL Ser358Asp (S358D) identified heat shock protein 90 (HSP90) as a high-confidence interacting protein. Further phenotypic characterization established MLKL as a novel HSP90 client. In summary, this novel inducible expression system enables SH-tag-based interaction studies in the cell line proficient for the respective phenotypic or signaling context and constitutes a valuable tool for experimental approaches requiring inducible or traceable protein expression.

  13. An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client*

    PubMed Central

    Bigenzahn, Johannes W.; Fauster, Astrid; Rebsamen, Manuele; Kandasamy, Richard K.; Scorzoni, Stefania; Vladimer, Gregory I.; Müller, André C.; Gstaiger, Matthias; Zuber, Johannes; Bennett, Keiryn L.; Superti-Furga, Giulio

    2016-01-01

    Tandem affinity purification–mass spectrometry (TAP-MS) is a popular strategy for the identification of protein–protein interactions, characterization of protein complexes, and entire networks. Its employment in cellular settings best fitting the relevant physiology is limited by convenient expression vector systems. We developed an easy-to-handle, inducible, dually selectable retroviral expression vector allowing dose- and time-dependent control of bait proteins bearing the efficient streptavidin-hemagglutinin (SH)-tag at their N- or C termini. Concomitant expression of a reporter fluorophore allows to monitor bait-expressing cells by flow cytometry or microscopy and enables high-throughput phenotypic assays. We used the system to successfully characterize the interactome of the neuroblastoma RAS viral oncogene homolog (NRAS) Gly12Asp (G12D) mutant and exploited the advantage of reporter fluorophore expression by tracking cytokine-independent cell growth using flow cytometry. Moreover, we tested the feasibility of studying cytotoxicity-mediating proteins with the vector system on the cell death-inducing mixed lineage kinase domain-like protein (MLKL) Ser358Asp (S358D) mutant. Interaction proteomics analysis of MLKL Ser358Asp (S358D) identified heat shock protein 90 (HSP90) as a high-confidence interacting protein. Further phenotypic characterization established MLKL as a novel HSP90 client. In summary, this novel inducible expression system enables SH-tag-based interaction studies in the cell line proficient for the respective phenotypic or signaling context and constitutes a valuable tool for experimental approaches requiring inducible or traceable protein expression. PMID:26933192

  14. Affinity membrane introduction mass spectrometry

    SciTech Connect

    Xu, C.; Patrick, J.S.; Cooks, R.G. )

    1995-02-15

    A new technique, affinity membrane introduction mass spectrometry, is described. In this method, a chemically modified membrane is used to selectively adsorb analytes bearing a particular functional group and concentrate them from solution. Release of the bound analyte results in its transfer across the membrane and allows it to be monitored mass spectrometrically, using, in the present case, a benchtop ion trap instrument. Alkylamine-modified cellulose membranes are used to bind substituted benzaldehydes through imine formation at high pH. Release of the bound aldehyde is achieved by acid hydrolysis of the surface-bound imine. Benzaldehyde is detected with excellent specificity at 10 ppm in a complex mixture using this method. Using the enrichment capability of the membrane, a full mass spectrum of benzaldehyde can be measured at a concentration of 10 ppb. The behavior of a variety of other aldehydes is also discussed to illustrate the capabilities of the method. 21 refs., 5 figs., 2 tabs.

  15. Targeting Synaptic Pathology with a Novel Affinity Mass Spectrometry Approach*

    PubMed Central

    Brinkmalm, Ann; Brinkmalm, Gunnar; Honer, William G.; Moreno, Julie A.; Jakobsson, Joel; Mallucci, Giovanna R.; Zetterberg, Henrik; Blennow, Kaj; Öhrfelt, Annika

    2014-01-01

    We report a novel strategy for studying synaptic pathology by concurrently measuring levels of four SNARE complex proteins from individual brain tissue samples. This method combines affinity purification and mass spectrometry and can be applied directly for studies of SNARE complex proteins in multiple species or modified to target other key elements in neuronal function. We use the technique to demonstrate altered levels of presynaptic proteins in Alzheimer disease patients and prion-infected mice. PMID:24973420

  16. SAINT-MS1: protein-protein interaction scoring using label-free intensity data in affinity purification – mass spectrometry experiments

    PubMed Central

    Choi, Hyungwon; Glatter, Timo; Gstaiger, Mathias; Nesvizhskii, Alexey I.

    2013-01-01

    We present a statistical method SAINT-MS1 for scoring protein-protein interactions based on the label-free MS1 intensity data from affinity purification - mass spectrometry (AP-MS) experiments. The method is an extension of Significance Analysis of INTeractome (SAINT), a model-based method previously developed for spectral count data. We reformulated the statistical model for the log-transformed intensity data, including adequate treatment of missing observations, i.e. interactions whose quantitative data are inconsistent over replicate purifications. We demonstrate the performance of SAINT-MS1 using two recently published datasets: a small LTQ-Orbitrap dataset with three replicate purifications of single human bait protein and control purifications, and a larger drosophila dataset targeting insulin receptor/target of rapamycin signaling pathway generated using an LTQ-FT instrument. Using the drosophila dataset, we also compare and discuss the performance of SAINT analysis based on spectral count and MS1 intensity data in terms of the recovery of orthologous and literature-curated interactions. Given rapid advances in high mass accuracy instrumentation and intensity-based label-free quantification software, we expect that SAINT-MS1 will become a useful tool allowing improved detection of protein interactions in label-free AP-MS data, especially in the low abundance range. PMID:22352807

  17. Parallel Exploration of Interaction Space by BioID and Affinity Purification Coupled to Mass Spectrometry.

    PubMed

    Hesketh, Geoffrey G; Youn, Ji-Young; Samavarchi-Tehrani, Payman; Raught, Brian; Gingras, Anne-Claude

    2017-01-01

    Complete understanding of cellular function requires knowledge of the composition and dynamics of protein interaction networks, the importance of which spans all molecular cell biology fields. Mass spectrometry-based proteomics approaches are instrumental in this process, with affinity purification coupled to mass spectrometry (AP-MS) now widely used for defining interaction landscapes. Traditional AP-MS methods are well suited to providing information regarding the temporal aspects of soluble protein-protein interactions, but the requirement to maintain protein-protein interactions during cell lysis and AP means that both weak-affinity interactions and spatial information is lost. A more recently developed method called BioID employs the expression of bait proteins fused to a nonspecific biotin ligase, BirA*, that induces in vivo biotinylation of proximal proteins. Coupling this method to biotin affinity enrichment and mass spectrometry negates many of the solubility and interaction strength issues inherent in traditional AP-MS methods, and provides unparalleled spatial context for protein interactions. Here we describe the parallel implementation of both BioID and FLAG AP-MS allowing simultaneous exploration of both spatial and temporal aspects of protein interaction networks.

  18. Identification of Evening Complex Associated Proteins in Arabidopsis by Affinity Purification and Mass Spectrometry*

    PubMed Central

    Shen, Zhouxin; Kay, Steve A.

    2016-01-01

    Many species possess an endogenous circadian clock to synchronize internal physiology with an oscillating external environment. In plants, the circadian clock coordinates growth, metabolism and development over daily and seasonal time scales. Many proteins in the circadian network form oscillating complexes that temporally regulate myriad processes, including signal transduction, transcription, protein degradation and post-translational modification. In Arabidopsis thaliana, a tripartite complex composed of EARLY FLOWERING 4 (ELF4), EARLY FLOWERING 3 (ELF3), and LUX ARRHYTHMO (LUX), named the evening complex, modulates daily rhythms in gene expression and growth through transcriptional regulation. However, little is known about the physical interactions that connect the circadian system to other pathways. We used affinity purification and mass spectrometry (AP-MS) methods to identify proteins that associate with the evening complex in A. thaliana. New connections within the circadian network as well as to light signaling pathways were identified, including linkages between the evening complex, TIMING OF CAB EXPRESSION1 (TOC1), TIME FOR COFFEE (TIC), all phytochromes and TANDEM ZINC KNUCKLE/PLUS3 (TZP). Coupling genetic mutation with affinity purifications tested the roles of phytochrome B (phyB), EARLY FLOWERING 4, and EARLY FLOWERING 3 as nodes connecting the evening complex to clock and light signaling pathways. These experiments establish a hierarchical association between pathways and indicate direct and indirect interactions. Specifically, the results suggested that EARLY FLOWERING 3 and phytochrome B act as hubs connecting the clock and red light signaling pathways. Finally, we characterized a clade of associated nuclear kinases that regulate circadian rhythms, growth, and flowering in A. thaliana. Coupling mass spectrometry and genetics is a powerful method to rapidly and directly identify novel components and connections within and between complex signaling

  19. Identification of Evening Complex Associated Proteins in Arabidopsis by Affinity Purification and Mass Spectrometry.

    PubMed

    Huang, He; Alvarez, Sophie; Bindbeutel, Rebecca; Shen, Zhouxin; Naldrett, Michael J; Evans, Bradley S; Briggs, Steven P; Hicks, Leslie M; Kay, Steve A; Nusinow, Dmitri A

    2016-01-01

    Many species possess an endogenous circadian clock to synchronize internal physiology with an oscillating external environment. In plants, the circadian clock coordinates growth, metabolism and development over daily and seasonal time scales. Many proteins in the circadian network form oscillating complexes that temporally regulate myriad processes, including signal transduction, transcription, protein degradation and post-translational modification. In Arabidopsis thaliana, a tripartite complex composed of EARLY FLOWERING 4 (ELF4), EARLY FLOWERING 3 (ELF3), and LUX ARRHYTHMO (LUX), named the evening complex, modulates daily rhythms in gene expression and growth through transcriptional regulation. However, little is known about the physical interactions that connect the circadian system to other pathways. We used affinity purification and mass spectrometry (AP-MS) methods to identify proteins that associate with the evening complex in A. thaliana. New connections within the circadian network as well as to light signaling pathways were identified, including linkages between the evening complex, TIMING OF CAB EXPRESSION1 (TOC1), TIME FOR COFFEE (TIC), all phytochromes and TANDEM ZINC KNUCKLE/PLUS3 (TZP). Coupling genetic mutation with affinity purifications tested the roles of phytochrome B (phyB), EARLY FLOWERING 4, and EARLY FLOWERING 3 as nodes connecting the evening complex to clock and light signaling pathways. These experiments establish a hierarchical association between pathways and indicate direct and indirect interactions. Specifically, the results suggested that EARLY FLOWERING 3 and phytochrome B act as hubs connecting the clock and red light signaling pathways. Finally, we characterized a clade of associated nuclear kinases that regulate circadian rhythms, growth, and flowering in A. thaliana. Coupling mass spectrometry and genetics is a powerful method to rapidly and directly identify novel components and connections within and between complex signaling

  20. Sodium Cation Affinities of Commonly Used MALDI Matrices Determined by Guided Ion Beam Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Chinthaka, S. D. M.; Rodgers, M. T.

    2012-04-01

    The sodium cation affinities of six commonly used MALDI matrices are determined here using guided ion beam tandem mass spectrometry techniques. The collision-induced dissociation behavior of six sodium cationized MALDI matrices, Na+(MALDI), with Xe is studied as a function of kinetic energy. The MALDI matrices examined here include: nicotinic acid, quinoline, 3-aminoquinoline, 4-nitroaniline, picolinic acid, and 3-hydroxypicolinic acid. In all cases, the primary dissociation pathway corresponds to endothermic loss of the intact MALDI matrix. The cross section thresholds are interpreted to yield zero and 298 K Na+-MALDI bond dissociation energies (BDEs), or sodium cation affinities, after accounting for the effects of multiple ion-neutral collisions, the kinetic and internal energy distributions of the reactants, and dissociation lifetimes. Density functional theory calculations at the B3LYP/6-311+G(2d,2p)//B3LYP/6-31G* and MP2(full)/6-311+G(2d,2p)//B3LYP/6-31G* levels of theory are used to characterized the structures and energetics for these systems. The calculated BDEs exhibit very good agreement with the measured values for most systems. The experimental and theoretical Na+-MALDI BDEs determined here are compared with those previously measured by cation transfer equilibrium methods.

  1. Affinity Selection-Mass Spectrometry Identifies a Novel Antibacterial RNA Polymerase Inhibitor.

    PubMed

    Walker, Scott S; Degen, David; Nickbarg, Elliott; Carr, Donna; Soriano, Aileen; Mandal, Mihir; Painter, Ronald E; Sheth, Payal; Xiao, Li; Sher, Xinwei; Murgolo, Nicholas; Su, Jing; Olsen, David B; Ebright, Richard H; Young, Katherine

    2017-03-31

    The growing prevalence of drug resistant bacteria is a significant global threat to human health. The antibacterial drug rifampin, which functions by inhibiting bacterial RNA polymerase (RNAP), is an important part of the antibacterial armamentarium. Here, in order to identify novel inhibitors of bacterial RNAP, we used affinity-selection mass spectrometry to screen a chemical library for compounds that bind to Escherichia coli RNAP. We identified a novel small molecule, MRL-436, that binds to RNAP, inhibits RNAP, and exhibits antibacterial activity. MRL-436 binds to RNAP through a binding site that differs from the rifampin binding site, inhibits rifampin-resistant RNAP derivatives, and exhibits antibacterial activity against rifampin-resistant strains. Isolation of mutants resistant to the antibacterial activity of MRL-436 yields a missense mutation in codon 622 of the rpoC gene encoding the RNAP β' subunit or a null mutation in the rpoZ gene encoding the RNAP ω subunit, confirming that RNAP is the functional cellular target for the antibacterial activity of MRL-436, and indicating that RNAP β' subunit residue 622 and the RNAP ω subunit are required for the antibacterial activity of MRL-436. Similarity between the resistance determinant for MRL-436 and the resistance determinant for the cellular alarmone ppGpp suggests a possible similarity in binding site and/or induced conformational state for MRL-436 and ppGpp.

  2. Epitope structure and binding affinity of single chain llama anti-β-amyloid antibodies revealed by proteolytic excision affinity-mass spectrometry.

    PubMed

    Paraschiv, Gabriela; Vincke, Cécile; Czaplewska, Paulina; Manea, Marilena; Muyldermans, Serge; Przybylski, Michael

    2013-01-01

    ß-Amyloid (Aß) immunotherapy has become a promising strategy for reducing the level of Aß in brain. New immunological approaches have been recently proposed for rapid, early diagnosis, and molecular treatment of neurodegenerative diseases related to Alzheimer's Disease (AD). The combination of proteolytic epitope excision and extraction and mass spectrometry using digestion with various proteases has been shown to be an efficient tool for the identification and molecular characterization of antigenic determinants. Here, we report the identification of the Aβ epitope recognized by the variable domain of single chain llama anti-Aβ-antibodies, termed Aβ-nanobodies, that have been discovered in the blood of camelids and found to be promising candidates for immunotherapy of AD. The epitope recognized by two Aβ-specific nanobodies was identified by proteolytic epitope extraction- and excision-mass spectrometry using a series of proteases (trypsin, chymotrypsin, GluC-protease, and LysC-protease). Matrix-assisted laser desorption ionization--mass spectrometric analysis of the affinity--elution fraction provided the epitope, Aβ(17-28), in the mid- to carboxy-terminal domain of Aβ, which has been shown to exert an Aß-fibril inhibiting effect. Affinity studies of the synthetic epitope confirmed that the Aβ(17-28) peptide is the minimal fragment that binds to the nanobodies. The interactions between the nanobodies and full length Aβ(1-40) or Aβ-peptides containing or lacking the epitope sequence were further characterized by enzyme linked immunosorbent assay and bioaffinity analysis. Determinations of binding affinities between the Aβ-nanobodies and Aβ(1-40) and the Aβ(17-28) epitope provided K(D) values of approximately 150 and 700 nmol, respectively. Thus, the knowledge of the epitope may be highly useful for future studies of Aβ-aggregation (oligomerization and fibril formation) and for designing new aggregation inhibitors.

  3. Comparative Study of Three Methods for Affinity Measurements: Capillary Electrophoresis Coupled with UV Detection and Mass Spectrometry, and Direct Infusion Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Mironov, Gleb G.; Logie, Jennifer; Okhonin, Victor; Renaud, Justin B.; Mayer, Paul M.; Berezovski, Maxim V.

    2012-07-01

    We present affinity capillary electrophoresis and mass spectrometry (ACE-MS) as a comprehensive separation technique for label-free solution-based affinity analysis. The application of ACE-MS for measuring affinity constants between eight small molecule drugs [ibuprofen, s-flurbiprofen, diclofenac, phenylbutazone, naproxen, folic acid, resveratrol, and 4,4'-(propane-1,3-diyl) dibenzoic acid] and β-cyclodextrin is described. We couple on-line ACE with MS to combine the separation and kinetic capability of ACE together with the molecular weight and structural elucidation of MS in one system. To understand the full potential of ACE-MS, we compare it with two other methods: Direct infusion mass spectrometry (DIMS) and ACE with UV detection (ACE-UV). After the evaluation, DIMS provides less reliable equilibrium dissociation constants than separation-based ACE-UV and ACE-MS, and cannot be used solely for the study of noncovalent interactions. ACE-MS determines apparent dissociation constants for all reacting small molecules in a mixture, even in cases when drugs overlap with each other during separation. The ability of ACE-MS to interact, separate, and rapidly scan through m/z can facilitate the simultaneous affinity analysis of multiple interacting pairs, potentially leading to the high-throughput screening of drug candidates.

  4. When is Mass Spectrometry Combined with Affinity Approaches Essential? A Case Study of Tyrosine Nitration in Proteins

    NASA Astrophysics Data System (ADS)

    Petre, Brînduşa-Alina; Ulrich, Martina; Stumbaum, Mihaela; Bernevic, Bogdan; Moise, Adrian; Döring, Gerd; Przybylski, Michael

    2012-11-01

    Tyrosine nitration in proteins occurs under physiologic conditions and is increased at disease conditions associated with oxidative stress, such as inflammation and Alzheimer's disease. Identification and quantification of tyrosine-nitrations are crucial for understanding nitration mechanism(s) and their functional consequences. Mass spectrometry (MS) is best suited to identify nitration sites, but is hampered by low stabilities and modification levels and possible structural changes induced by nitration. In this insight, we discuss methods for identifying and quantifying nitration sites by proteolytic affinity extraction using nitrotyrosine (NT)-specific antibodies, in combination with electrospray-MS. The efficiency of this approach is illustrated by identification of specific nitration sites in two proteins in eosinophil granules from several biological samples, eosinophil-cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). Affinity extraction combined with Edman sequencing enabled the quantification of nitration levels, which were found to be 8 % and 15 % for ECP and EDN, respectively. Structure modeling utilizing available crystal structures and affinity studies using synthetic NT-peptides suggest a tyrosine nitration sequence motif comprising positively charged residues in the vicinity of the NT- residue, located at specific surface- accessible sites of the protein structure. Affinities of Tyr-nitrated peptides from ECP and EDN to NT-antibodies, determined by online bioaffinity- MS, provided nanomolar KD values. In contrast, false-positive identifications of nitrations were obtained in proteins from cystic fibrosis patients upon using NT-specific antibodies, and were shown to be hydroxy-tyrosine modifications. These results demonstrate affinity- mass spectrometry approaches to be essential for unequivocal identification of biological tyrosine nitrations.

  5. Phosphopeptide screening using nanocrystalline titanium dioxide films as affinity matrix-assisted laser desorption ionization targets in mass spectrometry.

    PubMed

    Niklew, Marie-Luise; Hochkirch, Ulrike; Melikyan, Anna; Moritz, Thomas; Kurzawski, Sandra; Schlüter, Hartmut; Ebner, Ingo; Linscheid, Michael W

    2010-02-01

    The use of nanocrystalline titanium dioxide films as affinity targets for the selective isolation and enrichment of phosphopeptides with subsequent analysis by matrix-assisted laser desorption ionization (MALDI) mass spectrometry is described. A strong affinity of phosphopeptides to anatase titanium dioxide surfaces is observed, and a standard protocol for the selective isolation and enrichment of phosphopeptides on titanium dioxide films using a proteolytic digest of alpha- and beta-casein was developed. All washing and elution procedures using these films can be processed directly on the MALDI target, thereby avoiding sample contamination and losses. In addition, the enrichment of the phosphopeptides was improved due to a considerable enlargement of the surface. Several film substrates compatible with routine inlet systems of mass spectrometers, as conductive glass, aluminum, and silicon, have been manufactured and tested. A biological application was examined by the human fibrinogen-thrombin system. For a quantification and comparison of different expression levels of phosphoproteins in biological systems, the peptides were labeled with S-methyl thioimidate reagents. The capability of this method for high-throughput applications make the use of mesoporous titanium dioxide films as an affinity MALDI target a promising tool in phosphoproteomics. A combination of an amidation protocol showed that a quantification of phosphorylated peptides can easily be performed using TiO(2) films.

  6. Alkali-cation affinities of polyoxyethylene dodecylethers and helical conformations of their cationized molecules studied by electrospray mass spectrometry.

    PubMed

    Yokoyama, Yukio; Hirajima, Rui; Morigaki, Ken; Yamaguchi, Yoshitaka; Ueda, Kazuyoshi

    2007-11-01

    Relative alkali-cation affinity of polyoxyethylene (POE) dodecylethers in gas phase was studied by electrospray ionization (ESI) mass spectrometry using dodecylether-poly-ethoxylate (C(12)EO:n, "n" denotes ethyleneoxide unit number) nonionic surfactants, and possible helical conformations of the cationized molecules were demonstrated. The alkali-cation affinity highly depended on the cation diameters. The mass spectra of C(12)EO:8 cationized by alkali-metal ions were dominated by potassiated molecules. The results indicated that the POE moiety could have specific affinity to K(+) ions based on a host-guest interaction between POE helix and potassium ions. This is very similar to the relationships between 18-crown-6 and K(+). The ESI mass spectra exhibited the multiply cationized C(12)EO:n in addition to the singly cationized molecules. The critical EO unit numbers necessary for producing the multiply-charged cationized molecules also depended on the cation diameters. In addition, the POE surfactants highly preferred alkali cations to proton. The results were strongly supported by molecular mechanics/dynamics calculations. A helical conformation of the POE moiety of C(12)EO:15 including two K(+) ions gave a potential minimum, while a lowest energy structure of the protonated molecule took irregular conformations due to the formation of local hydrogen bonds.

  7. Identification of protein complexes in Escherichia coli using sequential peptide affinity purification in combination with tandem mass spectrometry.

    PubMed

    Babu, Mohan; Kagan, Olga; Guo, Hongbo; Greenblatt, Jack; Emili, Andrew

    2012-11-12

    Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems(1, 2). Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria(1-6). In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes(1, 2, 7). Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast(8, 9), and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system(10). Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large

  8. Recent advances in bacteria identification by matrix-assisted laser desorption/ionization mass spectrometry using nanomaterials as affinity probes.

    PubMed

    Chiu, Tai-Chia

    2014-04-28

    Identifying trace amounts of bacteria rapidly, accurately, selectively, and with high sensitivity is important to ensuring the safety of food and diagnosing infectious bacterial diseases. Microbial diseases constitute the major cause of death in many developing and developed countries of the world. The early detection of pathogenic bacteria is crucial in preventing, treating, and containing the spread of infections, and there is an urgent requirement for sensitive, specific, and accurate diagnostic tests. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an extremely selective and sensitive analytical tool that can be used to characterize different species of pathogenic bacteria. Various functionalized or unmodified nanomaterials can be used as affinity probes to capture and concentrate microorganisms. Recent developments in bacterial detection using nanomaterials-assisted MALDI-MS approaches are highlighted in this article. A comprehensive table listing MALDI-MS approaches for identifying pathogenic bacteria, categorized by the nanomaterials used, is provided.

  9. Recent Advances in Bacteria Identification by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Using Nanomaterials as Affinity Probes

    PubMed Central

    Chiu, Tai-Chia

    2014-01-01

    Identifying trace amounts of bacteria rapidly, accurately, selectively, and with high sensitivity is important to ensuring the safety of food and diagnosing infectious bacterial diseases. Microbial diseases constitute the major cause of death in many developing and developed countries of the world. The early detection of pathogenic bacteria is crucial in preventing, treating, and containing the spread of infections, and there is an urgent requirement for sensitive, specific, and accurate diagnostic tests. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an extremely selective and sensitive analytical tool that can be used to characterize different species of pathogenic bacteria. Various functionalized or unmodified nanomaterials can be used as affinity probes to capture and concentrate microorganisms. Recent developments in bacterial detection using nanomaterials-assisted MALDI-MS approaches are highlighted in this article. A comprehensive table listing MALDI-MS approaches for identifying pathogenic bacteria, categorized by the nanomaterials used, is provided. PMID:24786089

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

    PubMed

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

    2013-01-01

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

  11. Enrichment and Analysis of Nonenzymatically Glycated Peptides: Boronate Affinity Chromatography Coupled with Electron-Transfer Dissociation Mass Spectrometry

    SciTech Connect

    Zhang, Qibin; Tang, Ning; Brock, Jonathan W.; Mottaz, Heather M.; Ames, Jennifer M.; Baynes, John; Smith, Richard D.; Metz, Thomas O.

    2007-06-01

    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. However, no effective high-throughput methods exist for identifying proteins containing this low abundance post-translational modification in bottom-up proteomic studies. In this report, phenylboronate affinity chromatography was used in a two-step enrichment scheme to selectively isolate first glycated proteins and then glycated, tryptic peptides from human serum glycated in vitro. Enriched peptides were subsequently analyzed by alternating electron transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. It was observed that ETD fragmentation mode resulted in a significantly higher number of glycated peptide identifications (87.6% of all identified peptides) versus CID mode (17.0% of all identified peptides), when utilizing dual glycation enrichment on both the protein and peptide level. This study illustrates that phenylboronate affinity chromatography coupled with LC-MS/MS with ETD as the fragmentation mode is an efficient approach for analyses of glycated proteins and can have broad applications in studies of diabetes mellitus.

  12. Combined affinity labelling and mass spectrometry analysis of differential cell surface protein expression in normal and prostate cancer cells.

    PubMed

    Hastie, Claire; Saxton, Malcolm; Akpan, Akunna; Cramer, Rainer; Masters, John R; Naaby-Hansen, Soren

    2005-09-01

    Differences in the expression of cell surface proteins between a normal prostate epithelial (1542-NP2TX) and a prostate cancer cell line (1542-CP3TX) derived from the same patient were investigated. A combination of affinity chromatographic purification of biotin-tagged surface proteins with mass spectrometry analysis identified 26 integral membrane proteins and 14 peripheral surface proteins. The findings confirm earlier reports of altered expression in prostate cancer for several cell surface proteins, including ALCAM/CD166, the Ephrin type A receptor, EGFR and the prostaglandin F2 receptor regulatory protein. In addition, several novel findings of differential expression were made, including the voltage-dependent anion selective channel proteins Porin 1 and 2, ecto-5'-nucleotidase (CD73) and Scavenger receptor B1. Cell surface protein expression changed both qualitatively and quantitatively when the cells were grown in the presence of either or both interferon INFalpha and INFgamma. Costimulation with type I and II interferons had additive or synergistic effects on the membrane density of several, mainly peripherally attached surface proteins. Concerted upregulation of surface exposed antigens may be of benefit in immuno-adjuvant-based treatment of interferon-responsive prostate cancer. In conclusion, this study demonstrates that differences in the expression of membrane proteins between normal and prostate cancer cells are reproducibly detectable following vectorial labelling with biotin, and that detailed analysis of extracellular-induced surface changes can be achieved by combining surface-specific labelling with high-resolution two-dimensional gel electrophoresis and mass spectrometry.

  13. Substrate and Substrate-Mimetic Chaperone Binding Sites in Human α-Galactosidase A Revealed by Affinity-Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Moise, Adrian; Maeser, Stefan; Rawer, Stephan; Eggers, Frederike; Murphy, Mary; Bornheim, Jeff; Przybylski, Michael

    2016-06-01

    Fabry disease (FD) is a rare metabolic disorder of a group of lysosomal storage diseases, caused by deficiency or reduced activity of the enzyme α-galactosidase. Human α-galactosidase A (hαGAL) hydrolyses the terminal α-galactosyl moiety from glycosphingolipids, predominantly globotriaosylceramide (Gb3). Enzyme deficiency leads to incomplete or blocked breakdown and progressive accumulation of Gb3, with detrimental effects on normal organ functions. FD is successfully treated by enzyme replacement therapy (ERT) with purified recombinant hαGAL. An emerging treatment strategy, pharmacologic chaperone therapy (PCT), employs small molecules that can increase and/or reconstitute the activity of lysosomal enzyme trafficking by stabilizing misfolded isoforms. One such chaperone, 1-deoxygalactonojirimycin (DGJ), is a structural galactose analogue currently validated in clinical trials. DGJ is an active-site-chaperone that binds at the same or similar location as galactose; however, the molecular determination of chaperone binding sites in lysosomal enzymes represents a considerable challenge. Here we report the identification of the galactose and DGJ binding sites in recombinant α-galactosidase through a new affinity-mass spectrometry-based approach that employs selective proteolytic digestion of the enzyme-galactose or -inhibitor complex. Binding site peptides identified by mass spectrometry, [39-49], [83-100], and [141-168], contain the essential ligand-contacting amino acids, in agreement with the known X-ray crystal structures. The inhibitory effect of DGJ on galactose recognition was directly characterized through competitive binding experiments and mass spectrometry. The methods successfully employed in this study should have high potential for the characterization of (mutated) enzyme-substrate and -chaperone interactions, and for identifying chaperones without inhibitory effects.

  14. Using mass spectrometry to study the photo-affinity labeling of protein tyrosine phosphatase 1B

    NASA Astrophysics Data System (ADS)

    Leriche, Tammy; Skorey, Kathryn; Roy, Patrick; McKay, Dan; Bateman, Kevin P.

    2004-11-01

    Protein tyrosine phosphatase 1B (PTP1B) is a potential target for the treatment of Type II diabetes and several companies are developing small molecule inhibitors of this enzyme. Part of the characterization of these compounds as PTP1B inhibitors is the understanding of how they bind in the enzyme active site. The use of photo-activated inhibitors that target the active site can provide such insight. This paper describes the characterization of a photoprobe directed at the active site of PTP1B. Mass spectrometry revealed the specific binding of the probe to the intact protein. Digestion of the labeled protein followed by LC-MS and LC-MS/MS was used to show that the photoprobe binds to a specific active site amino acid. This was confirmed by comparison with the X-ray structure of PTP1B with a PTP1B inhibitor. The probe labels a conserved acidic residue (Asp) that is required for catalytic activity. This photoprobe may prove to be a useful tool for the development of a PTP1B inhibitor or for the study of PTPs in general.

  15. Monolith immuno-affinity enrichment liquid chromatography tandem mass spectrometry for quantitative protein analysis of recombinant bovine somatotropin in serum.

    PubMed

    Smits, Nathalie G E; Blokland, Marco H; Wubs, Klaas L; Nessen, Merel A; van Ginkel, Leen A; Nielen, Michel W F

    2015-08-01

    The use of recombinant bovine somatotropin (rbST) to enhance milk production is approved in several countries, but it is prohibited in the European Union. According to EU legislation, it is necessary to confirm positive screening results prior to enforcement. Although adequate screening assays are available nowadays, development of liquid chromatography tandem mass spectrometry (LC-MS/MS) confirmatory methods to detect low levels of rbST is still a challenge. Here, we present a novel approach using immuno-affinity enrichment on monolithic micro-columns in combination with state-of-the-art ultra-high pressure LC-MS/MS (UHPLC-MS/MS) detection. The developed approach enables detection and confirmation of rbST in serum at a decision limit (CCα) concentration of 0.8 ng mL(-1). Furthermore, the method is easy to handle, robust and reproducible. We successfully applied the confirmatory method to serum samples from rbST treated cows that were found suspect after immunoassay-based screening. The use of rbST could be confirmed over 1 week after treatment, and the developed method demonstrated the sensitivity needed for effective control. Graphical Abstract Graphical summary of the workflow, for serum preparation, enrichment with monolith microcolumns and LC-MS/MS measurement of rbST.

  16. Proteomic analysis of human O {sup 6}-methylguanine-DNA methyltransferase by affinity chromatography and tandem mass spectrometry

    SciTech Connect

    Niture, Suryakant K.; Doneanu, Catalin E.; Velu, Chinavenmeni S.; Bailey, Nathan I.; Srivenugopal, Kalkunte S. . E-mail: Kalkunte.srivenugopal@ttuhsc.edu

    2005-12-02

    Recent evidence suggests that human O {sup 6}-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein that protects the genome against mutagens and accords tumor resistance to many anticancer alkylating agents, may have other roles besides repair. Therefore, we isolated MGMT-interacting proteins from extracts of HT29 human colon cancer cells using affinity chromatography on MGMT-Sepharose. Specific proteins bound to this column were identified by electrospray ionization tandem mass spectrometry and/or Western blotting. These procedures identified >60 MGMT-interacting proteins with diverse functions including those involved in DNA replication and repair (MCM2, PCNA, ORC1, DNA polymerase {delta}, MSH-2, and DNA-dependent protein kinase), cell cycle progression (CDK1, cyclin B, CDK2, CDC7, CDC10, 14-3-3 protein, and p21{sup waf1/cip1}), RNA processing and translation (poly(A)-binding protein, nucleolin, heterogeneous nuclear ribonucleoproteins, A2/B1, and elongation factor-1{alpha}), several histones (H4, H3.4, and H2A.1), and topoisomerase I. The heat shock proteins, HSP-90{alpha} and {beta}, also bound strongly with MGMT. The DNA repair activity of MGMT was greatly enhanced in the presence of interacting proteins or histones. These data, for the first time, suggest that human MGMT is likely to have additional functions, possibly, in sensing and integrating the DNA damage/repair-related signals with replication, cell cycle progression, and genomic stability.

  17. Novel Chemical Ligands to Ebola Virus and Marburg Virus Nucleoproteins Identified by Combining Affinity Mass Spectrometry and Metabolomics Approaches

    PubMed Central

    Fu, Xu; Wang, Zhihua; Li, Lixin; Dong, Shishang; Li, Zhucui; Jiang, Zhenzuo; Wang, Yuefei; Shui, Wenqing

    2016-01-01

    The nucleoprotein (NP) of Ebola virus (EBOV) and Marburg virus (MARV) is an essential component of the viral ribonucleoprotein complex and significantly impacts replication and transcription of the viral RNA genome. Although NP is regarded as a promising antiviral druggable target, no chemical ligands have been reported to interact with EBOV NP or MARV NP. We identified two compounds from a traditional Chinese medicine Gancao (licorice root) that can bind both NPs by combining affinity mass spectrometry and metabolomics approaches. These two ligands, 18β-glycyrrhetinic acid and licochalcone A, were verified by defined compound mixture screens and further characterized with individual ligand binding assays. Accompanying biophysical analyses demonstrate that binding of 18β-glycyrrhetinic acid to EBOV NP significantly reduces protein thermal stability, induces formation of large NP oligomers, and disrupts the critical association of viral ssRNA with NP complexes whereas the compound showed no such activity on MARV NP. Our study has revealed the substantial potential of new analytical techniques in ligand discovery from natural herb resources. In addition, identification of a chemical ligand that influences the oligomeric state and RNA-binding function of EBOV NP sheds new light on antiviral drug development. PMID:27403722

  18. Identification and Affinity-Quantification of ß-Amyloid and α-Synuclein Polypeptides Using On-Line SAW-Biosensor-Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Slamnoiu, Stefan; Vlad, Camelia; Stumbaum, Mihaela; Moise, Adrian; Lindner, Kathrin; Engel, Nicole; Vilanova, Mar; Diaz, Mireia; Karreman, Christiaan; Leist, Marcel; Ciossek, Thomas; Hengerer, Bastian; Vilaseca, Marta; Przybylski, Michael

    2014-08-01

    Bioaffinity analysis using a variety of biosensors has become an established tool for detection and quantification of biomolecular interactions. Biosensors, however, are generally limited by the lack of chemical structure information of affinity-bound ligands. On-line bioaffinity-mass spectrometry using a surface-acoustic wave biosensor (SAW-MS) is a new combination providing the simultaneous affinity detection, quantification, and mass spectrometric structural characterization of ligands. We describe here an on-line SAW-MS combination for direct identification and affinity determination, using a new interface for MS of the affinity-isolated ligand eluate. Key element of the SAW-MS combination is a microfluidic interface that integrates affinity-isolation on a gold chip, in-situ sample concentration, and desalting with a microcolumn for MS of the ligand eluate from the biosensor. Suitable MS- acquisition software has been developed that provides coupling of the SAW-MS interface to a Bruker Daltonics ion trap-MS, FTICR-MS, and Waters Synapt-QTOF- MS systems. Applications are presented for mass spectrometric identifications and affinity (KD) determinations of the neurodegenerative polypeptides, ß-amyloid (Aß), and pathophysiological and physiological synucleins (α- and ß-synucleins), two key polypeptide systems for Alzheimer's disease and Parkinson's disease, respectively. Moreover, first in vivo applications of αSyn polypeptides from brain homogenate show the feasibility of on-line affinity-MS to the direct analysis of biological material. These results demonstrate on-line SAW-bioaffinity-MS as a powerful tool for structural and quantitative analysis of biopolymer interactions.

  19. An HLA-B27 Homodimer Specific Antibody Recognizes a Discontinuous Mixed-Disulfide Epitope as Identified by Affinity-Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Iuraşcu, Marius-Ionuţ; Marroquin Belaunzanar, Osiris; Cozma, Claudia; Petrausch, Ulf; Renner, Christoph; Przybylski, Michael

    2016-06-01

    HLA-B27 homodimer formation is believed to be a hallmark of HLA-B27 associated spondyloarthritides. Recently, we have generated a homodimer-specific monoclonal antibody (HD6) and have demonstrated that HLA-B27 homodimer complexes are present on monocytes of healthy HLA-B27 gene carriers at low levels, with significantly increased levels at active disease. The capability of the HD6 antibody to discriminate between correctly formed HLA-B27 heterotrimers and pathology-associated homodimers is striking and cannot be explained by the primary structure of HLA-B27. We hypothesized that HD6 accesses a unique epitope and used affinity-mass spectrometry for its identification. The HD6 antibody was immobilized on an activated sepharose affinity column, and HLA-B27 homodimer characterized for affinity. The epitope was identified by proteolytic epitope excision and MALDI mass spectrometry, and shown to comprise a discontinuous Cys-203- 257-Cys mixed-disulfide peptide structure that is not accessible in HLA-B27 heterotrimers due to protection by noncovalently linked β2-microglobulin. The epitope peptides were synthesized by solid phase peptide synthesis, and the two monomeric peptide components, HLA-B27(203-219) and HLA-B27(257-273), as well as the homo- and hetero-dimeric disulfide linked combinations prepared. The affinity binding constants KD towards the antibodies were determined using a surface acoustic wave (SAW) biosensor, and showed the highest affinity with a KD of approximately 40 nM to the HD6 antibody for the (203-219)-SS-(257-273) mixed disulfide epitope.

  20. Tandem Affinity Purification Approach Coupled to Mass Spectrometry to Identify Post-translational Modifications of Histones Associated with Chromatin-Binding Proteins.

    PubMed

    Beyer, Sophie; Robin, Philippe; Ait-Si-Ali, Slimane

    2017-01-01

    Protein purification by tandem affinity purification (TAP)-tag coupled to mass spectrometry analysis is usually used to reveal protein complex composition. Here we describe a TAP-tag purification of chromatin-bound proteins along with associated nucleosomes, which allow exhaustive identification of protein partners. Moreover, this method allows exhaustive identification of the post-translational modifications (PTMs) of the associated histones. Thus, in addition to partner characterization, this approach reveals the associated epigenetic landscape that can shed light on the function and properties of the studied chromatin-bound protein.

  1. Multiple ligand detection and affinity measurement by ultrafiltration and mass spectrometry analysis applied to fragment mixture screening.

    PubMed

    Qin, Shanshan; Ren, Yiran; Fu, Xu; Shen, Jie; Chen, Xin; Wang, Quan; Bi, Xin; Liu, Wenjing; Li, Lixin; Liang, Guangxin; Yang, Cheng; Shui, Wenqing

    2015-07-30

    Binding affinity of a small molecule drug candidate to a therapeutically relevant biomolecular target is regarded the first determinant of the candidate's efficacy. Although the ultrafiltration-LC/MS (UF-LC/MS) assay enables efficient ligand discovery for a specific target from a mixed pool of compounds, most previous analysis allowed for relative affinity ranking of different ligands. Moreover, the reliability of affinity measurement for multiple ligands with UF-LC/MS has hardly been strictly evaluated. In this study, we examined the accuracy of K(d) determination through UF-LC/MS by comparison with classical ITC measurement. A single-point K(d) calculation method was found to be suitable for affinity measurement of multiple ligands bound to the same target when binding competition is minimized. A second workflow based on analysis of the unbound fraction of compounds was then developed, which simplified sample preparation as well as warranted reliable ligand discovery. The new workflow implemented in a fragment mixture screen afforded rapid and sensitive detection of low-affinity ligands selectively bound to the RNA polymerase NS5B of hepatitis C virus. More importantly, ligand identification and affinity measurement for mixture-based fragment screens by UF-LC/MS were in good accordance with single ligand evaluation by conventional SPR analysis. This new approach is expected to become a valuable addition to the arsenal of high-throughput screening techniques for fragment-based drug discovery.

  2. Profiling of drug binding proteins by monolithic affinity chromatography in combination with liquid chromatography-tandem mass spectrometry.

    PubMed

    Zhang, Xuepei; Wang, Tongdan; Zhang, Hanzhi; Han, Bing; Wang, Lishun; Kang, Jingwu

    2014-09-12

    A new approach for proteome-wide profiling drug binding proteins by using monolithic capillary affinity chromatography in combination with HPLC-MS/MS is reported. Two immunosuppresive drugs, namely FK506 and cyclosporin A, were utilized as the experimental models for proof-of-concept. The monolithic capillary affinity columns were prepared through a single-step copolymerization of the drug derivatives with glycidyl methacrylate and ethylene dimethacrylate. The capillary chromatography with the affinity monolithic column facilitates the purification of the drug binding proteins from the cell lysate. By combining the capillary affinity column purification and the shot-gun proteomic analysis, totally 33 FK506- and 32 CsA-binding proteins including all the literature reported target proteins of these two drugs were identified. Among them, two proteins, namely voltage-dependent anion-selective channel protein 1 and serine/threonine-protein phosphatase PGAM5 were verified by using the recombinant proteins. The result supports that the monolithic capillary affinity chromatography is likely to become a valuable tool for profiling of binding proteins of small molecular drugs as well as bioactive compounds.

  3. Identification of differential protein binding affinities in an atropisomeric pharmaceutical compound by noncovalent mass spectrometry, equilibrium dialysis, and nuclear magnetic resonance.

    PubMed

    Maple, Hannah J; Garlish, Rachel A; Whitcombe, Ian; Hold, Adam; Prosser, Christine E; Ford, Daniel; Mackenzie, Harry; Crosby, John; Porter, John; Taylor, Richard J; Crump, Matthew P

    2013-06-18

    Atropisomerism of pharmaceutical compounds is a challenging area for drug discovery programs (Angew. Chem., Int. Ed. 2009, 48, 6398-6401). Strategies for dealing with these compounds include raising the energy barrier to atropisomerization in order to develop the drug as a single isomer (Tetrahedron 2004, 60, 4337-4347) or reducing the barrier to rotation and developing a mixture of rapidly interconverting isomers (Chirality 1996, 8, 364-371). Commonly, however, the atropisomers will be differentiated in terms of their affinity for a given protein target, and it is therefore important to rapidly identify the most active component prior to further compound development. We present equilibrium dialysis and saturation transfer difference NMR (STD-NMR) as techniques for assessing relative affinities of an atropisomeric mixture against antiapoptotic protein targets Bcl-2 and Bcl-xL. These techniques require no prior separation of the mixture of compounds and are therefore rapid and simple approaches. We also explore the use of noncovalent mass spectrometry for determining KD values of individual atropisomers separated from the equilibrium mixture and compare the results to solution-phase measurements. Results from equilibrium dialysis, STD-NMR, and noncovalent mass spectrometry are all in excellent agreement and provide complementary information on differential binding, amplification of the strongest binders, and KD values.

  4. Tandem affinity purification of histones, coupled to mass spectrometry, identifies associated proteins and new sites of post-translational modification in Saccharomyces cerevisiae.

    PubMed

    Valero, M Luz; Sendra, Ramon; Pamblanco, Mercè

    2016-03-16

    Histones and their post-translational modifications contribute to regulating fundamental biological processes in all eukaryotic cells. We have applied a conventional tandem affinity purification strategy to histones H3 and H4 of the yeast Saccharomyces cerevisiae. Mass spectrometry analysis of the co-purified proteins revealed multiple associated proteins, including core histones, which indicates that tagged histones may be incorporated to the nucleosome particle. Among the many other co-isolated proteins there are histone chaperones, elements of chromatin remodeling, of nucleosome assembly/disassembly, and of histone modification complexes. The histone chaperone Rtt106p, two members of chromatin assembly FACT complex and Psh1p, an ubiquitin ligase, were the most abundant proteins obtained with both H3-TAP and H4-TAP, regardless of the cell extraction medium stringency. Our mass spectrometry analyses have also revealed numerous novel post-translational modifications, including 30 new chemical modifications in histones, mainly by ubiquitination. We have discovered not only new sites of ubiquitination but that, besides lysine, also serine and threonine residues are targets of ubiquitination on yeast histones. Our results show the standard tandem affinity purification procedure is suitable for application to yeast histones, in order to isolate and characterize histone-binding proteins and post-translational modifications, avoiding the bias caused by histone purification from a chromatin-enriched fraction.

  5. Application of high-throughput affinity-selection mass spectrometry for screening of chemical compound libraries in lead discovery.

    PubMed

    Zehender, Hartmut; Mayr, Lorenz M

    2007-02-01

    High-throughput screening of chemical libraries for compounds that interfere with a particular molecular target is among the most powerful methodologies applied in lead discovery at present. In this review, the authors describe a label-free, homogeneous, affinity-selection-based technology developed at Novartis, termed SpeedScreen, which is compared with similar technologies used for high-throughput screening in the pharmaceutical and biotechnology industries. The focus at present of SpeedScreen is twofold: first, this technology is applied to orphan genomic targets and to those targets that are non-tractable by a functional assay; second, this technology is applied complementary to the well-established traditional methodologies for the screening of molecular targets. In summary, the authors discuss the value of affinity-selection-based high-throughput screening as a complementary technology to the common functional screening platforms and the benefits as well as the limitations of this new technology are outlined.

  6. Improved accuracy of low affinity protein-ligand equilibrium dissociation constants directly determined by electrospray ionization mass spectrometry.

    PubMed

    Jaquillard, Lucie; Saab, Fabienne; Schoentgen, Françoise; Cadene, Martine

    2012-05-01

    There is continued interest in the determination by ESI-MS of equilibrium dissociation constants (K(D)) that accurately reflect the affinity of a protein-ligand complex in solution. Issues in the measurement of K(D) are compounded in the case of low affinity complexes. Here we present a K(D) measurement method and corresponding mathematical model dealing with both gas-phase dissociation (GPD) and aggregation. To this end, a rational mathematical correction of GPD (f(sat)) is combined with the development of an experimental protocol to deal with gas-phase aggregation. A guide to apply the method to noncovalent protein-ligand systems according to their kinetic behavior is provided. The approach is validated by comparing the K(D) values determined by this method with in-solution K(D) literature values. The influence of the type of molecular interactions and instrumental setup on f(sat) is examined as a first step towards a fine dissection of factors affecting GPD. The method can be reliably applied to a wide array of low affinity systems without the need for a reference ligand or protein.

  7. Cryptic antigenic determinants on the extracellular pyruvate dehydrogenase complex/mimeotope found in primary biliary cirrhosis. A probe by affinity mass spectrometry.

    PubMed

    Yip, T T; Van de Water, J; Gershwin, M E; Coppel, R L; Hutchens, T W

    1996-12-20

    Affinity mass spectrometry (AMS) was used to evaluate the structural diversity of the E2 component of pyruvate dehydrogenase complex (PDC) in normal and diseased liver cells, including those from patients with the autoimmune disease primary biliary cirrhosis (PBC). Two different antibodies to PDC-E2, the immunodominant mitochondrial autoantigen in patients with PBC, were used. AMS was performed directly on frozen liver sections and purified bile duct epithelial cells. Mass spectrometric signals associated with the molecular recognition of PBC-specific antigenic determinants were enhanced by an in situ enzyme-linked signal amplification process. Samples from patients with PBC gave strong positive signals for the antigen(s) recognized by the monoclonal antibody C355.1. Conversely, tissues from normal and disease controls showed only a minimal signal. AMS was used to identify specific antigenic determinants within the E2 component of PDC for comparison with unknown antigenic determinants observed by affinity capture with C355.1 monoclonal antibody from PBC samples. PDC components bound to C355.1 were mapped and identified by mass before dissociation from the E2 component. A similar approach was used to identify unknown antigenic determinants associated with PBC. We believe AMS may be an important new approach with wide application to the identification of molecules associated with a number of disease states.

  8. A method to resolve the composition of heterogeneous affinity-purified protein complexes assembled around a common protein by chemical cross-linking, gel electrophoresis and mass spectrometry.

    PubMed

    Rudashevskaya, Elena L; Sacco, Roberto; Kratochwill, Klaus; Huber, Marie L; Gstaiger, Matthias; Superti-Furga, Giulio; Bennett, Keiryn L

    2013-01-01

    Protein complexes form, dissociate and re-form in order to perform specific cellular functions. In this two-pronged protocol, noncovalent protein complexes are initially isolated by affinity purification for subsequent identification of the components by liquid chromatography high-resolution mass spectrometry (LC-MS) on a hybrid LTQ Orbitrap Velos. In the second prong of the approach, the affinity-purification strategy includes a chemical cross-linking step to 'freeze' a series of concurrently formed, heterogeneous protein subcomplex species that are visualized by gel electrophoresis. This branch of the methodology amalgamates standard and well-practiced laboratory methods to reveal compositional changes that occur in protein complex architecture. By using mouse N-terminally tagged streptavidin-binding peptide-hemagglutinin-TANK-binding kinase 1 (SH-TBK1), we chemically cross-linked the affinity-purified complex of SH-TBK1 with the homobifunctional lysine-specific reagent bis(sulfosuccinimidyl) suberate (BS(3)), and we separated the resultant protein complexes by denaturation and by silver-stained one- and two-dimensional SDS-PAGE. We observed a range of cross-linked TBK1 complexes of variable pI and M(r) and confirmed them by immunoblotting. LC-MS analysis of in situ-digested cross-linked proteins shows differences in the composition of the TBK1 subcomplexes. The protocol is inherently simple and can be readily extended to the investigation of a range of protein complexes. From cell lysis to data generation by LC-MS, the protocol takes approximately 2.5 to 5.5 d to perform.

  9. Detection and identification of heme c-modified peptides by histidine affinity chromatography, high-performance liquid chromatography-mass spectrometry, and database searching.

    PubMed

    Merkley, Eric D; Anderson, Brian J; Park, Jea; Belchik, Sara M; Shi, Liang; Monroe, Matthew E; Smith, Richard D; Lipton, Mary S

    2012-12-07

    Multiheme c-type cytochromes (proteins with covalently attached heme c moieties) play important roles in extracellular metal respiration in dissimilatory metal-reducing bacteria. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) characterization of c-type cytochromes is hindered by the presence of multiple heme groups, since the heme c modified peptides are typically not observed or, if observed, not identified. Using a recently reported histidine affinity chromatography (HAC) procedure, we enriched heme c tryptic peptides from purified bovine heart cytochrome c, two bacterial decaheme cytochromes, and subjected these samples to LC-MS/MS analysis. Enriched bovine cytochrome c samples yielded 3- to 6-fold more confident peptide-spectrum matches to heme c containing peptides than unenriched digests. In unenriched digests of the decaheme cytochrome MtoA from Sideroxydans lithotrophicus ES-1, heme c peptides for 4 of the 10 expected sites were observed by LC-MS/MS; following HAC fractionation, peptides covering 9 out of 10 sites were obtained. Heme c peptide spiked into E. coli lysates at mass ratios as low as 1×10(-4) was detected with good signal-to-noise after HAC and LC-MS/MS analysis. In addition to HAC, we have developed a proteomics database search strategy that takes into account the unique physicochemical properties of heme c peptides. The results suggest that accounting for the double thioether link between heme c and peptide, and the use of the labile heme fragment as a reporter ion, can improve database searching results. The combination of affinity chromatography and heme-specific informatics yielded increases in the number of peptide-spectrum matches of 20-100-fold for bovine cytochrome c.

  10. Affinity capture of biotinylated proteins at acidic conditions to facilitate hydrogen/deuterium exchange mass spectrometry analysis of multimeric protein complexes.

    PubMed

    Jensen, Pernille Foged; Jørgensen, Thomas J D; Koefoed, Klaus; Nygaard, Frank; Sen, Jette Wagtberg

    2013-08-06

    Characterization of conformational and dynamic changes associated with protein interactions can be done by hydrogen/deuterium exchange mass spectrometry (HDX-MS) by comparing the deuterium uptake in the bound and unbound state of the proteins. Investigation of local hydrogen/deuterium exchange in heteromultimeric protein complexes poses a challenge for the method due to the increased complexity of the mixture of peptides originating from all interaction partners in the complex. Previously, interference of peptides from one interaction partner has been removed by immobilizing the intact protein on beads prior to the HDX-MS experiment. However, when studying protein complexes of more than two proteins, immobilization can possibly introduce steric limitations to the interactions. Here, we present a method based on the high affinity biotin-streptavidin interaction that allows selective capture of biotinylated proteins even under the extreme conditions for hydrogen/deuterium exchange quenching i.e. pH 2.5 and 0 °C. This biotin-streptavidin capture strategy allows hydrogen/deuterium exchange to occur in proteins in solution and enables characterization of specific proteins in heteromultimeric protein complexes without interference of peptides originating from other interaction partners in the complex. The biotin-streptavidin strategy has been successfully implemented in a model system with two recombinant monoclonal antibodies that target nonoverlapping epitopes on the human epidermal growth factor receptor (EGFR). We present a workflow for biotinylation and characterization of recombinant antibodies and demonstrate affinity capture of biotinylated antibodies under hydrogen/deuterium exchange quench conditions by the biotin-streptavidin strategy.

  11. Detection and Identification of Heme c-Modified Peptides by Histidine Affinity Chromatography, High-Performance Liquid Chromatography-Mass Spectrometry, and Database Searching

    SciTech Connect

    Merkley, Eric D.; Anderson, Brian J.; Park, Jea H.; Belchik, Sara M.; Shi, Liang; Monroe, Matthew E.; Smith, Richard D.; Lipton, Mary S.

    2012-12-07

    Multiheme c-type cytochromes (proteins with covalently attached heme c moieties) play important roles in extracellular metal respiration in dissimilatory metal-reducing bacteria. Liquid chromatography-tandem mass spectrometry-(LC-MS/MS) characterization of c-type cytochromes is hindered by the presence of multiple heme groups, since the heme c modified peptides are typically not observed, or if observed, not identified. Using a recently reported histidine affinity chromatography (HAC) procedure, we enriched heme c tryptic peptides from purified bovine heart cytochrome c, a bacterial decaheme cytochrome, and subjected these samples to LC-MS/MS analysis. Enriched bovine cytochrome c samples yielded three- to six-fold more confident peptide-spectrum matches to heme-c containing peptides than unenriched digests. In unenriched digests of the decaheme cytochrome MtoA from Sideroxydans lithotrophicus ES-1, heme c peptides for four of the ten expected sites were observed by LC-MS/MS; following HAC fractionation, peptides covering nine out of ten sites were obtained. Heme c peptide spiked into E. coli lysates at mass ratios as low as 10-4 was detected with good signal-to-noise after HAC and LC-MS/MS analysis. In addition to HAC, we have developed a proteomics database search strategy that takes into account the unique physicochemical properties of heme c peptides. The results suggest that accounting for the double thioether link between heme c and peptide, and the use of the labile heme fragment as a reporter ion, can improve database searching results. The combination of affinity chromatography and heme-specific informatics yielded increases in the number of peptide-spectrum matches of 20-100-fold for bovine cytochrome c.

  12. Characterization of the novel broad-spectrum kinase inhibitor CTx-0294885 as an affinity reagent for mass spectrometry-based kinome profiling.

    PubMed

    Zhang, Luxi; Holmes, Ian P; Hochgräfe, Falko; Walker, Scott R; Ali, Naveid A; Humphrey, Emily S; Wu, Jianmin; de Silva, Melanie; Kersten, Wilhelmus J A; Connor, Theresa; Falk, Hendrik; Allan, Lynda; Street, Ian P; Bentley, John D; Pilling, Patricia A; Monahan, Brendon J; Peat, Thomas S; Daly, Roger J

    2013-07-05

    Kinase enrichment utilizing broad-spectrum kinase inhibitors enables the identification of large proportions of the expressed kinome by mass spectrometry. However, the existing inhibitors are still inadequate in covering the entire kinome. Here, we identified a novel bisanilino pyrimidine, CTx-0294885, exhibiting inhibitory activity against a broad range of kinases in vitro, and further developed it into a Sepharose-supported kinase capture reagent. Use of a quantitative proteomics approach confirmed the selectivity of CTx-0294885-bound beads for kinase enrichment. Large-scale CTx-0294885-based affinity purification followed by LC-MS/MS led to the identification of 235 protein kinases from MDA-MB-231 cells, including all members of the AKT family that had not been previously detected by other broad-spectrum kinase inhibitors. Addition of CTx-0294885 to a mixture of three kinase inhibitors commonly used for kinase-enrichment increased the number of kinase identifications to 261, representing the largest kinome coverage from a single cell line reported to date. Coupling phosphopeptide enrichment with affinity purification using the four inhibitors enabled the identification of 799 high-confidence phosphosites on 183 kinases, ∼10% of which were localized to the activation loop, and included previously unreported phosphosites on BMP2K, MELK, HIPK2, and PRKDC. Therefore, CTx-0294885 represents a powerful new reagent for analysis of kinome signaling networks that may facilitate development of targeted therapeutic strategies. Proteomics data have been deposited to the ProteomeXchange Consortium ( http://proteomecentral.proteomexchange.org ) via the PRIDE partner repository with the data set identifier PXD000239.

  13. Development of a Magnetic Microbead Affinity Selection Screen (MagMASS) Using Mass Spectrometry for Ligands to the Retinoid X Receptor-α

    NASA Astrophysics Data System (ADS)

    Rush, Michael D.; Walker, Elisabeth M.; Prehna, Gerd; Burton, Tristesse; van Breemen, Richard B.

    2016-12-01

    To overcome limiting factors in mass spectrometry-based screening methods such as automation while still facilitating the screening of complex mixtures such as botanical extracts, magnetic microbead affinity selection screening (MagMASS) was developed. The screening process involves immobilization of a target protein on a magnetic microbead using a variety of possible chemistries, incubation with mixtures of molecules containing possible ligands, a washing step that removes non-bound compounds while a magnetic field retains the beads in the microtiter well, and an organic solvent release step followed by LC-MS analysis. Using retinoid X receptor-α (RXRα) as an example, which is a nuclear receptor and target for anti-inflammation therapy as well as cancer treatment and prevention, a MagMASS assay was developed and compared with an existing screening assay, pulsed ultrafiltration (PUF)-MS. Optimization of MagMASS involved evaluation of multiple protein constructs and several magnetic bead immobilization chemistries. The full-length RXRα construct immobilized with amylose beads provided optimum results. Additional enhancements of MagMASS were the application of 96-well plates to enable automation, use of UHPLC instead of HPLC for faster MS analyses, and application of metabolomics software for faster, automated data analysis. Performance of MagMASS was demonstrated using mixtures of synthetic compounds and known ligands spiked into botanical extracts.

  14. Development of a Magnetic Microbead Affinity Selection Screen (MagMASS) Using Mass Spectrometry for Ligands to the Retinoid X Receptor-α

    NASA Astrophysics Data System (ADS)

    Rush, Michael D.; Walker, Elisabeth M.; Prehna, Gerd; Burton, Tristesse; van Breemen, Richard B.

    2017-03-01

    To overcome limiting factors in mass spectrometry-based screening methods such as automation while still facilitating the screening of complex mixtures such as botanical extracts, magnetic microbead affinity selection screening (MagMASS) was developed. The screening process involves immobilization of a target protein on a magnetic microbead using a variety of possible chemistries, incubation with mixtures of molecules containing possible ligands, a washing step that removes non-bound compounds while a magnetic field retains the beads in the microtiter well, and an organic solvent release step followed by LC-MS analysis. Using retinoid X receptor-α (RXRα) as an example, which is a nuclear receptor and target for anti-inflammation therapy as well as cancer treatment and prevention, a MagMASS assay was developed and compared with an existing screening assay, pulsed ultrafiltration (PUF)-MS. Optimization of MagMASS involved evaluation of multiple protein constructs and several magnetic bead immobilization chemistries. The full-length RXRα construct immobilized with amylose beads provided optimum results. Additional enhancements of MagMASS were the application of 96-well plates to enable automation, use of UHPLC instead of HPLC for faster MS analyses, and application of metabolomics software for faster, automated data analysis. Performance of MagMASS was demonstrated using mixtures of synthetic compounds and known ligands spiked into botanical extracts.

  15. Direct analysis of the products of sequential cleavages of peptides and proteins affinity-bound to immobilized metal ion beads by matrix-assisted laser desorption/ionization mass spectrometry.

    PubMed

    Qian, X; Zhou, W; Khaledi, M G; Tomer, K B

    1999-10-15

    Consecutive enzymatic reactions on analytes affinity-bound to immobilized metal ion beads with subsequent direct analysis of the products by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry have been used for detecting protein synthesis errors occuring at the N-terminus. The usefulness of this method was demonstrated by analyzing two commercially available recombinant HIV proteins with affinity tags at the N-terminus, and histatin-5, a peptide with multiple histidine residues. The high specificity, sensitivity, and speed of analysis make this method especially useful in obtaining N-terminal sequencing information of histidine-tagged recombinant proteins.

  16. Simultaneous speciation of selenoproteins and selenometabolites in plasma and serum by dual size exclusion-affinity chromatography with online isotope dilution inductively coupled plasma mass spectrometry.

    PubMed

    García-Sevillano, M A; García-Barrera, T; Gómez-Ariza, J L

    2014-04-01

    A method for the simultaneous speciation of selenoproteins and selenometabolites in mouse plasma has been developed based on in series two-dimensional size exclusion and affinity high-performance liquid chromatography (2D/SE-AF-HPLC), using two columns of each type, and hyphenation to inductively coupled plasma-(quadrupole) mass spectrometry (ICP-QMS). The method allows the quantitative determination of selenoprotein P (SeP), extracellular glutathione peroxidase (eGPx), selenoalbumin (SeAlb), and selenometabolites in mouse plasma using species-unspecific isotope dilution (SUID). The 2D chromatographic separation is proposed to remove typical spectral interferences in plasma from chloride and bromide on (77)Se ((40)Ar(37)Cl) and (82)Se ((81)Br(1)H). In addition, the approach increases chromatographic resolution allowing the separation of eGPx from Se metabolites of low molecular mass. The method is robust, reliable, and fast with a typical chromatographic runtime less than 20 min. Precision in terms of relative standard deviation (n = 5) is in the order of 4 %, and detection limits are in the range of 0.2 to 1.0 ng Se g(-1). Method accuracy for determination of total protein bound to Se was assessed by analyzing human serum reference material (BCR-637) certified for total Se content, and latterly applied to mouse plasma (Mus musculus). In summary, a reliable speciation method for the analysis of eGPx, selenometabolites, SeP, and SeAlb in plasma/serum samples is proposed for the first time and is applicable to the evaluation of Se status in human in clinical studies and other mammals for environmental or toxicological assessment.

  17. Evidence for phosphorylation of serine 753 in CFTR using a novel metal-ion affinity resin and matrix-assisted laser desorption mass spectrometry.

    PubMed Central

    Neville, D. C.; Rozanas, C. R.; Price, E. M.; Gruis, D. B.; Verkman, A. S.; Townsend, R. R.

    1997-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes an apical membrane Cl- channel regulated by protein phosphorylation. To identify cAMP-dependent protein kinase (PKA)-phosphorylated residues in full-length CFTR, immobilized metal-ion affinity chromatography (IMAC) was used to selectively purify phosphopeptides. The greater specificity of iron-loaded (Fe3+) nitrilotriacetic (NTA). Sepharose compared to iminodiacetic acid (IDA) metal-chelating matrices was demonstrated using a PKA-phosphorylated recombinant NBD1-R protein from CFTR. Fe(3+)-loaded NTA Sepharose preferentially bound phosphopeptides, whereas acidic and poly-His-containing peptides were co-purified using the conventional IDA matrices. IMAC using NTA Sepharose enabled the selective recovery of phosphopeptides and identification of phosphorylated residues from a complex proteolytic digest. Phosphopeptides from PKA-phosphorylated full-length CFTR, generated in Hi5 insect cells using a baculovirus expression system, were purified using NTA Sepharose. Phosphopeptides were identified using matrix-assisted laser desorption mass spectrometry (MALDI/MS) with post-source decay (PSD) analysis and collision-induced dissociation (CID) experiments. Phosphorylated peptides were identified by mass and by the metastable loss of HPO3 and H3PO4 from the parent ions. Peptide sequence and phosphorylation at CFTR residues 660Ser, 737Ser, and 795Ser were confirmed using MALDI/PSD analysis. Peptide sequences and phosphorylation at CFTR residues 700Ser, 712Ser, 768Ser, and 813Ser were deduced from peptide mass, metastable fragment ion formation, and PKA consensus sequences. Peptide sequence and phosphorylation at residue 753Ser was confirmed using MALDI/CID analysis. This is the first report of phosphorylation of 753Ser in full-length CFTR. PMID:9385646

  18. Simultaneous high-throughput determination of interaction kinetics for drugs and cyclodextrins by high performance affinity chromatography with mass spectrometry detection.

    PubMed

    Wang, Caifen; Wang, Xiaobo; Xu, Xiaonan; Liu, Botao; Xu, Xu; Sun, Lixin; Li, Haiyan; Zhang, Jiwen

    2016-02-25

    The individual determination of the apparent dissociation rate constant (kd,app) using high performance affinity chromatography (HPAC) is a tedious process requiring numerous separate tests and massive data fitting, unable to provide the apparent association rate constant (ka) and equilibrium binding constant (Ka). In this study, a HPAC with mass spectrometry detection (HPAC-MS/MS) was employed to determine the drug-cyclodextrin (CD) interaction kinetics with low sample loading quantity (<10 ng per injection for single compound) and high-throughput yield as twenty drugs determined in one injection. The kd,app measured by HPAC-MS/MS approach were 0.89 ± 0.07, 4.34 ± 0.01, 1.48 ± 0.01 and 7.77 ± 0.04 s(-1) for ketoprofen, trimethoprim, indapamide and acetaminophen, with kd,app for acetaminophen consistent with that from the HPAC method with UV detector in our previous studies. For twenty drugs with diverse structures and chemical properties, good correlationship was found between kd,app measured by single compound analysis method and high-throughput HPAC-MS/MS approach, with the correlation coefficient of 0.987 and the significance F less than 0.001. Comprehensive quantification of ka,app, kd,app and Ka values was further performed based on the measurement of kd,app by peak profiling method and Ka by the peak fitting method. And the investigation of the drug-CD interaction kinetics under different conditions indicated that the column temperature and mobile phase composition significantly affected the determination of ka,app, kd,app and Ka while also dependent on the acidity and basicity of drugs. In summary, the high-throughput HPAC-MS/MS approach has been demonstrated high efficiency in determination of the drug-CD primary interaction kinetic parameter, especially, kd,app, being proven as a novel tool in screening the right CD for the solubilization of the right drug.

  19. Comprehensive profiling of ribonucleosides modification by affinity zirconium oxide-silica composite monolithic column online solid-phase microextraction - Mass spectrometry analysis.

    PubMed

    Jiang, Han-Peng; Chu, Jie-Mei; Lan, Meng-Dan; Liu, Ping; Yang, Na; Zheng, Fang; Yuan, Bi-Feng; Feng, Yu-Qi

    2016-09-02

    More than 140 modified ribonucleosides have been identified in RNA. Determination of endogenous modified ribonucleosides in biological fluids may serve as non-invasive disease diagnostic strategy. However, detection of the modified ribonucleosides in biological fluids is challenging, especially for the low abundant modified ribonucleosides due to the serious matrix interferences of biological fluids. Here, we developed a facile preparation strategy and successfully synthesized zirconium oxide-silica (ZrO2/SiO2) composite capillary monolithic column that exhibited excellent performance for the selective enrichment of cis-diol-containing compounds. Compared with the boronate-based affinity monolith, the ZrO2/SiO2 monolith showed ∼2 orders of magnitude higher extraction capacity and can be used under physiological pH (pH 6.5-7.5). Using the prepared ZrO2/SiO2 composite monolith as the trapping column and reversed-phase C18 column as the analytical column, we further established an online solid-phase microextraction (SPME) in combination with liquid chromatography-mass spectrometry (online SPME-LC-MS/MS) analysis for the comprehensive profiling of ribonucleosides modification in human urine. Our results showed that 68 cis-diol-containing ribosylated compounds were identified in human urine, which is, to the best of our knowledge, the highest numbers of cis-diol-containing compounds were determined in a single analysis. It is worth noting that four modified ribonucleosides were discovered in the human urine for the first time. In addition, the quantification results from the pooled urine samples showed that compared to healthy controls, the contents of sixteen ribose conjugates in the urine of gastric cancer, eleven in esophagus cancer and seven in lymphoma increased more than two folds. Among these ribose conjugates, four ribose conjugates increased more than two folds in both gastric cancer and esophagus cancer; three ribose conjugates increased more than two

  20. Ultra-fast liquid chromatography with tandem mass spectrometry determination of ochratoxin A in traditional Chinese medicines based on vortex-assisted solid-liquid microextraction and aptamer-affinity column clean-up.

    PubMed

    Yang, Xihui; Hu, Yichen; Kong, Weijun; Chu, Xianfeng; Yang, Meihua; Zhao, Ming; Ouyang, Zhen

    2014-11-01

    A rapid, selective, and sensitive ultra-fast liquid chromatography with tandem mass spectrometry method was developed for the determination of ochratoxin A in traditional Chinese medicines based on vortex-assisted solid-liquid microextraction and aptamer-affinity column clean-up. Through optimizing the sample pretreatment procedures and chromatographic conditions, good linearity (r(2) ≥ 0.9993), low limit of detection (0.5-0.8 μg/kg), and satisfactory recovery (83.54-94.44%) expressed the good reliability and applicability of the established method in various traditional Chinese medicines. Moreover, the aptamer-affinity column, prepared in-house, showed an excellent feasibility owing to its specific identification of ochratoxin A in various kinds of selected traditional Chinese medicines. The maximum adsorption amount and applicability value were 188.96 ± 10.56 ng and 72.3%, respectively. The matrix effects were effectively eliminated, especially for m/z 404.2→358.0 of ochratoxin A. The application of the developed method for screening the natural contamination levels of ochratoxin A in 25 random traditional Chinese medicines on the market in China indicated that only eight samples were contaminated with low levels below the legal limit (5.0 μg/kg) set by the European Union. This study provided a preferred choice for the rapid and accurate monitoring of ochratoxin A in complex matrices.

  1. HAMS: High-Affinity Mass Spectrometry Screening. A High-Throughput Screening Method for Identifying the Tightest-Binding Lead Compounds for Target Proteins with No False Positive Identifications

    NASA Astrophysics Data System (ADS)

    Imaduwage, Kasun P.; Go, Eden P.; Zhu, Zhikai; Desaire, Heather

    2016-11-01

    A major challenge in drug discovery is the identification of high affinity lead compounds that bind a particular target protein; these leads are typically identified by high throughput screens. Mass spectrometry has become a detection method of choice in drug screening assays because the target and the ligand need not be modified. Label-free assays are advantageous because they can be developed more rapidly than assays requiring labels, and they eliminate the risk of the label interfering with the binding event. However, in commonly used MS-based screening methods, detection of false positives is a major challenge. Here, we describe a detection strategy designed to eliminate false positives. In this approach, the protein and the ligands are incubated together, and the non-binders are separated for detection. Hits (protein binders) are not detectable by MS after incubation with the protein, but readily identifiable by MS when the target protein is not present in the incubation media. The assay was demonstrated using three different proteins and hundreds of non-inhibitors; no false positive hits were identified in any experiment. The assay can be tuned to select for ligands of a particular binding affinity by varying the quantity of protein used and the immobilization method. As examples, the method selectively detected inhibitors that have Ki values of 0.2 μM, 50 pM, and 700 pM. These findings demonstrate that the approach described here compares favorably with traditional MS-based screening methods.

  2. HAMS: High-Affinity Mass Spectrometry Screening. A High-Throughput Screening Method for Identifying the Tightest-Binding Lead Compounds for Target Proteins with No False Positive Identifications.

    PubMed

    Imaduwage, Kasun P; Go, Eden P; Zhu, Zhikai; Desaire, Heather

    2016-11-01

    A major challenge in drug discovery is the identification of high affinity lead compounds that bind a particular target protein; these leads are typically identified by high throughput screens. Mass spectrometry has become a detection method of choice in drug screening assays because the target and the ligand need not be modified. Label-free assays are advantageous because they can be developed more rapidly than assays requiring labels, and they eliminate the risk of the label interfering with the binding event. However, in commonly used MS-based screening methods, detection of false positives is a major challenge. Here, we describe a detection strategy designed to eliminate false positives. In this approach, the protein and the ligands are incubated together, and the non-binders are separated for detection. Hits (protein binders) are not detectable by MS after incubation with the protein, but readily identifiable by MS when the target protein is not present in the incubation media. The assay was demonstrated using three different proteins and hundreds of non-inhibitors; no false positive hits were identified in any experiment. The assay can be tuned to select for ligands of a particular binding affinity by varying the quantity of protein used and the immobilization method. As examples, the method selectively detected inhibitors that have Ki values of 0.2 μM, 50 pM, and 700 pM. These findings demonstrate that the approach described here compares favorably with traditional MS-based screening methods. Graphical Abstract ᅟ.

  3. An affinity selection-mass spectrometry method for the identification of small molecule ligands from self-encoded combinatorial libraries: Discovery of a novel antagonist of E. coli dihydrofolate reductase

    NASA Astrophysics Data System (ADS)

    Annis, D. Allen; Athanasopoulos, John; Curran, Patrick J.; Felsch, Jason S.; Kalghatgi, Krishna; Lee, William H.; Nash, Huw M.; Orminati, Jean-Paul A.; Rosner, Kristin E.; Shipps, Gerald W., Jr.; Thaddupathy, G. R. A.; Tyler, Andrew N.; Vilenchik, Lev; Wagner, Carston R.; Wintner, Edward A.

    2004-11-01

    The NeoGenesis Automated Ligand Identification System (ALIS), an affinity selection-mass spectrometry (AS-MS) process consisting of a rapid size-exclusion chromatography stage integrated with reverse-phase chromatography, electrospray mass spectrometry, and novel data searching algorithms, was used to screen mass-encoded, 2500-member combinatorial libraries, leading to the discovery of a novel, bioactive ligand for the anti-infective target Escherichia coli dihydrofolate reductase (DHFR). Synthesis of the mass-encoded, ligand-containing library, discussion of the deconvolution process for verifying the structure of the ligand through independent synthesis and screening in a small mixture (sub-library) format, and ALIS-MS/MS techniques to assign its regioisomeric connectivity are presented. ALIS-based competition experiments between the newly discovered ligand and other, known DHFR ligands, and biological activity assessments with stereo- and regioisomers of the hit compound confirm its DHFR-specific biological activity. The method described requires no foreknowledge of the structure or biochemistry of the protein target, consumes less than 1 [mu]g protein to screen >2500 compounds in a single experiment, and enables screening of >250,000 compounds per system per day. These advantages highlight the potential of the ALIS method for drug discovery against genomic targets with unknown biological function, as well as validated targets for which traditional discovery efforts have failed.

  4. Identification of phosphoproteins in Arabidopsis thaliana leaves using polyethylene glycol fractionation, immobilized metal-ion affinity chromatography, two-dimensional gel electrophoresis and mass spectrometry.

    PubMed

    Aryal, Uma K; Krochko, Joan E; Ross, Andrew R S

    2012-01-01

    Reversible protein phosphorylation is a key regulatory mechanism in cells. Identification and characterization of phosphoproteins requires specialized enrichment methods, due to the relatively low abundance of these proteins, and is further complicated in plants by the high abundance of Rubisco in green tissues. We present a novel method for plant phosphoproteome analysis that depletes Rubisco using polyethylene glycol fractionation and utilizes immobilized metal-ion affinity chromatography to enrich phosphoproteins. Subsequent protein separation by one- and two-dimensional gel electrophoresis is further improved by extracting the PEG-fractionated protein samples with SDS/phenol and methanol/chloroform to remove interfering compounds. Using this approach, we identified 132 phosphorylated proteins in a partial Arabidopsis leaf extract. These proteins are involved in a range of biological processes, including CO(2) fixation, protein assembly and folding, stress response, redox regulation, and cellular metabolism. Both large and small subunits of Rubisco were phosphorylated at multiple sites, and depletion of Rubisco enhanced detection of less abundant phosphoproteins, including those associated with state transitions between photosystems I and II. The discovery of a phosphorylated form of AtGRP7, a self-regulating RNA-binding protein that affects floral transition, as well as several previously uncharacterized ribosomal proteins confirm the utility of this approach for phosphoproteome analysis and its potential to increase our understanding of growth and development in plants.

  5. High throughput screening of high-affinity ligands for proteins with anion-binding sites using desorption electrospray ionization (DESI) mass spectrometry.

    PubMed

    Lu, Xin; Ning, Baoming; He, Dacheng; Huang, Lingyun; Yue, Xiangjun; Zhang, Qiming; Huang, Haiwei; Liu, Yang; He, Lan; Ouyang, Jin

    2014-03-01

    A high throughput screening system involving a linear ion trap (LTQ) analyzer, a house-made platform and a desorption electrospray ionization (DESI) source was established to screen ligands with a high affinity for proteins with anion-binding sites. The complexes were analyzed after incubation, ultrafiltration, washing, and displacement. A new anionic region inhibited dissociation (ARID) mechanism that was suitable for a protein with anion-binding site was proposed. We utilized the differences in detectable dissociation of protein-ligand complexes, combined with displacement experiments, to distinguish free ligands displaced from anion-binding sites from liberated ligands dissociated from nonspecific interactions. The method was validated by α1-acid glycoprotein (AGP) and (R), (S)-amlodipine. Site-specific enantioselectivity shown in our experiments was consistent with earlier studies. Obtaining all of the qualitative information of 15*3 samples in 2.3 min indicates that the analysis process is no longer the time-limiting step in the initial stage of drug discovery. Quantitative information verified that our method was at least a semiquantitative method.

  6. High Throughput Screening of High-Affinity Ligands for Proteins with Anion-Binding Sites using Desorption Electrospray Ionization (DESI) Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Lu, Xin; Ning, Baoming; He, Dacheng; Huang, Lingyun; Yue, Xiangjun; Zhang, Qiming; Huang, Haiwei; Liu, Yang; He, Lan; Ouyang, Jin

    2014-03-01

    A high throughput screening system involving a linear ion trap (LTQ) analyzer, a house-made platform and a desorption electrospray ionization (DESI) source was established to screen ligands with a high affinity for proteins with anion-binding sites. The complexes were analyzed after incubation, ultrafiltration, washing, and displacement. A new anionic region inhibited dissociation (ARID) mechanism that was suitable for a protein with anion-binding site was proposed. We utilized the differences in detectable dissociation of protein-ligand complexes, combined with displacement experiments, to distinguish free ligands displaced from anion-binding sites from liberated ligands dissociated from nonspecific interactions. The method was validated by α1-acid glycoprotein (AGP) and (R), (S)-amlodipine. Site-specific enantioselectivity shown in our experiments was consistent with earlier studies. Obtaining all of the qualitative information of 15*3 samples in 2.3 min indicates that the analysis process is no longer the time-limiting step in the initial stage of drug discovery. Quantitative information verified that our method was at least a semiquantitative method.

  7. Novel cartilage oligomeric matrix protein (COMP) neoepitopes identified in synovial fluids from patients with joint diseases using affinity chromatography and mass spectrometry.

    PubMed

    Åhrman, Emma; Lorenzo, Pilar; Holmgren, Kristin; Grodzinsky, Alan J; Dahlberg, Leif E; Saxne, Tore; Heinegård, Dick; Önnerfjord, Patrik

    2014-07-25

    To identify patients at risk for progressive joint damage, there is a need for early diagnostic tools to detect molecular events leading to cartilage destruction. Isolation and characterization of distinct cartilage oligomeric matrix protein (COMP) fragments derived from cartilage and released into synovial fluid will allow discrimination between different pathological conditions and monitoring of disease progression. Early detection of disease and processes in the tissue as well as an understanding of the pathologic mechanisms will also open the way for novel treatment strategies. Disease-specific COMP fragments were isolated by affinity chromatography of synovial fluids from patients with rheumatoid arthritis, osteoarthritis, or acute trauma. Enriched COMP fragments were separated by SDSPAGE followed by in-gel digestion and mass spectrometric identification and characterization.Using the enzymes trypsin, chymotrypsin, and Asp-N for the digestions, an extensive analysis of the enriched fragments could be accomplished. Twelve different neoepitopes were identified and characterized within the enriched COMP fragments. For one of the neoepitopes, Ser77, an inhibition ELISA was developed. This ELISA quantifies COMP fragments clearly distinguishable from total COMP. Furthermore, fragments containing the neoepitope Ser77 were released into the culture medium of cytokine (TNF-α and IL-6/soluble IL-6 receptor)-stimulated human cartilage explants. The identified neoepitopes provide a complement to the currently available commercial assays for cartilage markers. Through neoepitope assays, tools to pinpoint disease progression, evaluation methods for therapy, and means to elucidate disease mechanisms will be provided.

  8. Novel Cartilage Oligomeric Matrix Protein (COMP) Neoepitopes Identified in Synovial Fluids from Patients with Joint Diseases Using Affinity Chromatography and Mass Spectrometry*

    PubMed Central

    Åhrman, Emma; Lorenzo, Pilar; Holmgren, Kristin; Grodzinsky, Alan J.; Dahlberg, Leif E.; Saxne, Tore; Heinegård, Dick; Önnerfjord, Patrik

    2014-01-01

    To identify patients at risk for progressive joint damage, there is a need for early diagnostic tools to detect molecular events leading to cartilage destruction. Isolation and characterization of distinct cartilage oligomeric matrix protein (COMP) fragments derived from cartilage and released into synovial fluid will allow discrimination between different pathological conditions and monitoring of disease progression. Early detection of disease and processes in the tissue as well as an understanding of the pathologic mechanisms will also open the way for novel treatment strategies. Disease-specific COMP fragments were isolated by affinity chromatography of synovial fluids from patients with rheumatoid arthritis, osteoarthritis, or acute trauma. Enriched COMP fragments were separated by SDS-PAGE followed by in-gel digestion and mass spectrometric identification and characterization. Using the enzymes trypsin, chymotrypsin, and Asp-N for the digestions, an extensive analysis of the enriched fragments could be accomplished. Twelve different neoepitopes were identified and characterized within the enriched COMP fragments. For one of the neoepitopes, Ser77, an inhibition ELISA was developed. This ELISA quantifies COMP fragments clearly distinguishable from total COMP. Furthermore, fragments containing the neoepitope Ser77 were released into the culture medium of cytokine (TNF-α and IL-6/soluble IL-6 receptor)-stimulated human cartilage explants. The identified neoepitopes provide a complement to the currently available commercial assays for cartilage markers. Through neoepitope assays, tools to pinpoint disease progression, evaluation methods for therapy, and means to elucidate disease mechanisms will be provided. PMID:24917676

  9. [Analysis of rice leaves proteomes by liquid chromatography-tandem, mass spectrometry based on the purification using a novel affinity detergent removal spin column].

    PubMed

    Cao, Xiaolin; Gong, Jiadi; Chen, Mingxue; Yu, Shasha; Bian, Yingfang; Cao, Zhaoyun

    2014-11-01

    A purification method was established for the analysis of proteomes in rice leaves based on a novel detergent removal spin column (DRSC). The proteins were extracted by phenol protein extraction method followed by sodium dodecyl sulfate (SDS) lysis. The lysate was purified by the detergent removal spin column and the enzymolytic peptides were detected by the nanoflow liquid chromatography-hybrid linear trap quadrupole orbitrap mass spectrometry (nanoLC-LTQ/Orbitrap). In terms of SDS removal efficiencies and protein identification, the method of DRSC was compared with those of filter aided sample preparation (FASP) and acetone precipitation. As a result, there were good efficiencies ( > 95%) of SDS removal for the three methods. With the DRSC purification strategy, 563 proteins were identified from rice leaves, while only 196 and 306 proteins were identified by FASP and acetone precipitation procedures respectively, in spite of certain complementarities among these identified proteins by the three methods. DRSC is suitable for proteins with various relative molecular masses and pI values. However, there were similar losses of proteins with different relative molecular masses and pI values with the other two methods. Using the established method, 588 proteins were identified by once injection analysis. According to the molecular functions, 296 proteins with at least two identified peptides can be classified into eight categories with binding activity, enzyme activity, transporter activity, inhibitor activity, structural constitute, catalytic activity, other and unknown functions. The method provides technical reference for conducting rice proteomes.

  10. Profiling of cis-Diol-containing Nucleosides and Ribosylated Metabolites by Boronate-affinity Organic-silica Hybrid Monolithic Capillary Liquid Chromatography/Mass Spectrometry

    PubMed Central

    Jiang, Han-Peng; Qi, Chu-Bo; Chu, Jie-Mei; Yuan, Bi-Feng; Feng, Yu-Qi

    2015-01-01

    RNA contains a large number of modified nucleosides. In the metabolic re-exchange of RNA, modified nucleosides cannot be recycled and are thus excreted from cells into biological fluids. Determination of endogenous modified nucleosides in biological fluids may serve as non-invasive cancers diagnostic methods. Here we prepared boronate-affinity organic-silica hybrid capillary monolithic column (BOHCMC) that exhibited excellent selectivity toward the cis-diol-containing compounds. We then used the prepared BOHCMC as the on-line solid-phase microextraction (SPME) column and developed an on-line SPME-LC-MS/MS method to comprehensively profile cis-diol-containing nucleosides and ribosylated metabolites in human urine. Forty-five cis-diol-containing nucleosides and ribosylated metabolites were successfully identified in human urine. And five ribose conjugates, for the first time, were identified existence in human urine in the current study. Furthermore, the relative quantification suggested 4 cis-diol-containing compounds (5′-deoxy-5′-methylthioadensine, N4-acetylcytidine, 1-ribosyl-N-propionylhistamine and N2,N2,7-trimethylguanosine) increased more than 1.5 folds in all the 3 types of examined cancers (lung cancer, colorectal cancer, and nasopharyngeal cancer) compared to healthy controls. The on-line SPME-LC-MS/MS method demonstrates a promising method for the comprehensive profiling of cis-diol-containing ribose conjugates in human urines, which provides an efficient strategy for the identification and discovery of biomarkers and may be used for the screening of cancers. PMID:25585609

  11. Popular computational methods to assess multiprotein complexes derived from label-free affinity purification and mass spectrometry (AP-MS) experiments.

    PubMed

    Armean, Irina M; Lilley, Kathryn S; Trotter, Matthew W B

    2013-01-01

    Advances in sensitivity, resolution, mass accuracy, and throughput have considerably increased the number of protein identifications made via mass spectrometry. Despite these advances, state-of-the-art experimental methods for the study of protein-protein interactions yield more candidate interactions than may be expected biologically owing to biases and limitations in the experimental methodology. In silico methods, which distinguish between true and false interactions, have been developed and applied successfully to reduce the number of false positive results yielded by physical interaction assays. Such methods may be grouped according to: (1) the type of data used: methods based on experiment-specific measurements (e.g., spectral counts or identification scores) versus methods that extract knowledge encoded in external annotations (e.g., public interaction and functional categorisation databases); (2) the type of algorithm applied: the statistical description and estimation of physical protein properties versus predictive supervised machine learning or text-mining algorithms; (3) the type of protein relation evaluated: direct (binary) interaction of two proteins in a cocomplex versus probability of any functional relationship between two proteins (e.g., co-occurrence in a pathway, sub cellular compartment); and (4) initial motivation: elucidation of experimental data by evaluation versus prediction of novel protein-protein interaction, to be experimentally validated a posteriori. This work reviews several popular computational scoring methods and software platforms for protein-protein interactions evaluation according to their methodology, comparative strengths and weaknesses, data representation, accessibility, and availability. The scoring methods and platforms described include: CompPASS, SAINT, Decontaminator, MINT, IntAct, STRING, and FunCoup. References to related work are provided throughout in order to provide a concise but thorough introduction to a

  12. Quantification of the IgG2/4 kappa Monoclonal Therapeutic Eculizumab from Serum Using Isotype Specific Affinity Purification and Microflow LC-ESI-Q-TOF Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Ladwig, Paula M.; Barnidge, David R.; Willrich, Maria A. V.

    2016-12-01

    As therapeutic monoclonal antibodies (mAbs) become more humanized, traditional tryptic peptide approaches used to measure biologics in serum become more challenging since unique clonotypic peptides used for quantifying the mAb may also be found in the normal serum polyclonal background. An alternative approach is to monitor the unique molecular mass of the intact light chain portion of the mAbs using liquid chromatography-mass spectrometry (LC-MS). Distinguishing a therapeutic mAb from a patient's normal polyclonal immunoglobulin (Ig) repertoire is the primary limiting factor when determining the limit of quantitation (LOQ) in serum. The ability to selectively extract subclass specific Igs from serum reduces the polyclonal background in a sample. We present here the development of an LC-MS method to quantify eculizumab in serum. Eculizumab is a complement component 5 (C5) binding mAb that is fully humanized and contains portions of both IgG2 and IgG4 subclasses. Our group developed a method that uses Life Technologies CaptureSelect IgG4 (Hu) affinity matrix. We show here the ability to quantitate eculizumab with a LOQ of 5 mcg/mL by removing the higher abundance IgG1, IgG2, and IgG3 from the polyclonal background, making this approach a simple and efficient procedure.

  13. Continuous affinity-gradient nano-stationary phase served as a column for reversed-phase electrochromatography and matrix carrier in time-of-flight mass spectrometry for protein analysis.

    PubMed

    Wu, Jen-Kuei; Yang, Chung-Shi; Wu, Yi-Shiuan; Wang, Pen-Cheng; Tseng, Fan-Gang

    2015-08-19

    This study developed an affinity-gradient nano-stationary phase (AG-NSP) for protein analysis using nanofluidic capillary electrochromatography (nano-CEC) conjugated with matrix assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). The AG-NSP can be used for protein pre-separation in nano-CEC and as a matrix carrier for protein analysis in MALDI-TOF-MS. A hydrophobicity gradient in AG-NSP was photochemically formed by grafting 4-azidoaniline hydrochloride on vertically arrayed multi-wall carbon nanotubes (MWCNTs) through gray-level exposure to UV light. The reversed-phase gradient stationary phase in AG-NSP was tailored according to the properties of the mobile phase gradient in capillary electrochromatography. As a result, the operation of the system is easily automated using a single buffer solution without the need for multiple solvents for elution. The use of nano-CEC with AG-NSP demonstrated excellent separation efficiency and high resolution for various types of DNA/protein/peptide. MALDI-TOF-MS analysis was then performed directly on the separated proteins and peptides on the chip. The proposed system was then used for the detection of three types of proteins with different molecular weights and PI values, including Cytochrome c (12,360, pI = 10), Lysozyme (14,300, pI = 11), and BSA (86,000, pI = 5)), and digested IgG fragments. The proposed system provided resolution of 1000 Da for the proteins in this study and the separation of digested IgG fragments at a low concentration of 1.2 pmol μL(-1).

  14. Proteomic analysis of nipple aspirate fluid from women with early-stage breast cancer using isotope-coded affinity tags and tandem mass spectrometry reveals differential expression of vitamin D binding protein

    PubMed Central

    Pawlik, Timothy M; Hawke, David H; Liu, Yanna; Krishnamurthy, Savitri; Fritsche, Herbert; Hunt, Kelly K; Kuerer, Henry M

    2006-01-01

    Background Isotope-coded affinity tag (ICAT) tandem mass spectrometry (MS) allows for qualitative and quantitative analysis of paired protein samples. We sought to determine whether ICAT technology could quantify and identify differential expression of tumor-specific proteins in nipple aspirate fluid (NAF) from the tumor-bearing and contralateral disease-free breasts of patients with unilateral early-stage breast cancer. Methods Paired NAF samples from 18 women with stage I or II unilateral invasive breast carcinoma and 4 healthy volunteers were analyzed using ICAT labeling, sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), liquid chromatography, and MS. Proteins were identified by sequence database analysis. Western blot analysis of NAF from an independent sample set from 12 women (8 with early-stage breast cancer and 4 healthy volunteers) was also performed. Results 353 peptides were identified from tandem mass spectra and matched to peptide sequences in the National Center for Biotechnology Information database. Equal numbers of peptides were up- versus down-regulated. Alpha2HS-glycoprotein [Heavy:Light (H:L) ratio 0.63] was underexpressed in NAF from tumor-bearing breasts, while lipophilin B (H:L ratio 1.42), beta-globin (H:L ratio 1.98), hemopexin (H:L ratio 1.73), and vitamin D-binding protein precursor (H:L ratio 1.82) were overexpressed. Western blot analysis of pooled samples of NAF from healthy volunteers versus NAF from women with breast cancer confirmed the overexpression of vitamin D-binding protein in tumor-bearing breasts. Conclusion ICAT tandem MS was able to identify and quantify differences in specific protein expression between NAF samples from tumor-bearing and disease-free breasts. Proteomic screening techniques using ICAT and NAF may be used to find markers for diagnosis of breast cancer. PMID:16542425

  15. Discovery and characterization of EIIB, a new α-conotoxin from Conus ermineus venom by nAChRs affinity capture monitored by MALDI-TOF/TOF mass spectrometry.

    PubMed

    Echterbille, Julien; Gilles, Nicolas; Araóz, Romulo; Mourier, Gilles; Amar, Muriel; Servent, Denis; De Pauw, Edwin; Quinton, Loic

    2017-05-01

    Animal toxins are peptides that often bind with remarkable affinity and selectivity to membrane receptors such as nicotinic acetylcholine receptors (nAChRs). The latter are, for example, targeted by α-conotoxins, a family of peptide toxins produced by venomous cone snails. nAChRs are implicated in numerous physiological processes explaining why the design of new pharmacological tools and the discovery of potential innovative drugs targeting these receptor channels appear so important. This work describes a methodology developed to discover new ligands of nAChRs from complex mixtures of peptides. The methodology was set up by the incubation of Torpedo marmorata electrocyte membranes rich in nAChRs with BSA tryptic digests (>100 peptides) doped by small amounts of known nAChRs ligands (α-conotoxins). Peptides that bind to the receptors were purified and analyzed by MALDI-TOF/TOF mass spectrometry which revealed an enrichment of α-conotoxins in membrane-containing fractions. This result exhibits the binding of α-conotoxins to nAChRs. Negative controls were performed to demonstrate the specificity of the binding. The usefulness and the power of the methodology were also investigated for a discovery issue. The workflow was then applied to the screening of Conus ermineus crude venom, aiming at characterizing new nAChRs ligands from this venom, which has not been extensively investigated to date. The methodology validated our experiments by allowing us to bind two α-conotoxins (α-EI and α-EIIA) which have already been described as nAChRs ligands. Moreover, a new conotoxin, never described to date, was also captured, identified and sequenced from this venom. Classical pharmacology tests by radioligand binding using a synthetic homologue of the toxin confirm the activity of the new peptide, called α-EIIB. The Ki value of this peptide for Torpedo nicotinic receptors was measured at 2.2 ± 0.7 nM.

  16. A dielectric affinity microbiosensor

    NASA Astrophysics Data System (ADS)

    Huang, Xian; Li, Siqi; Schultz, Jerome S.; Wang, Qian; Lin, Qiao

    2010-01-01

    We present an affinity biosensing approach that exploits changes in dielectric properties of a polymer due to its specific, reversible binding with an analyte. The approach is demonstrated using a microsensor comprising a pair of thin-film capacitive electrodes sandwiching a solution of poly(acrylamide-ran-3-acrylamidophenylboronic acid), a synthetic polymer with specific affinity to glucose. Binding with glucose induces changes in the permittivity of the polymer, which can be measured capacitively for specific glucose detection, as confirmed by experimental results at physiologically relevant concentrations. The dielectric affinity biosensing approach holds the potential for practical applications such as long-term continuous glucose monitoring.

  17. Affinity in electrophoresis.

    PubMed

    Heegaard, Niels H H

    2009-06-01

    The journal Electrophoresis has greatly influenced my approaches to biomolecular affinity studies. The methods that I have chosen as my main tools to study interacting biomolecules--native gel and later capillary zone electrophoresis--have been the topic of numerous articles in Electrophoresis. Below, the role of the journal in the development and dissemination of these techniques and applications reviewed. Many exhaustive reviews on affinity electrophoresis and affinity CE have been published in the last few years and are not in any way replaced by the present deliberations that are focused on papers published by the journal.

  18. Quantitative analysis of low-abundance serological proteins with peptide affinity-based enrichment and pseudo-multiple reaction monitoring by hybrid quadrupole time-of-flight mass spectrometry.

    PubMed

    Kim, Kwang Hoe; Ahn, Yeong Hee; Ji, Eun Sun; Lee, Ju Yeon; Kim, Jin Young; An, Hyun Joo; Yoo, Jong Shin

    2015-07-02

    Multiple reaction monitoring (MRM) is commonly used for the quantitative analysis of proteins during mass pectrometry (MS), and has excellent specificity and sensitivity for an analyte in a complex sample. In this study, a pseudo-MRM method for the quantitative analysis of low-abundance serological proteins was developed using hybrid quadrupole time-of-flight (hybrid Q-TOF) MS and peptide affinity-based enrichment. First, a pseudo-MRM-based analysis using hybrid Q-TOF MS was performed for synthetic peptides selected as targets and spiked into tryptic digests of human serum. By integrating multiple transition signals corresponding to fragment ions in the full scan MS/MS spectrum of a precursor ion of the target peptide, a pseudo-MRM MS analysis of the target peptide showed an increased signal-to-noise (S/N) ratio and sensitivity, as well as an improved reproducibility. The pseudo-MRM method was then used for the quantitative analysis of the tryptic peptides of two low-abundance serological proteins, tissue inhibitor of metalloproteinase 1 (TIMP1) and tissue-type protein tyrosine phosphatase kappa (PTPκ), which were prepared with peptide affinity-based enrichment from human serum. Finally, this method was used to detect femtomolar amounts of target peptides derived from TIMP1 and PTPκ, with good coefficients of variation (CV 2.7% and 9.8%, respectively), using a few microliters of human serum from colorectal cancer patients. The results suggest that pseudo-MRM using hybrid Q-TOF MS, combined with peptide affinity-based enrichment, could become a promising alternative for the quantitative analysis of low-abundance target proteins of interest in complex serum samples that avoids protein depletion.

  19. Affine dynamics with torsion

    NASA Astrophysics Data System (ADS)

    Gültekin, Kemal

    2016-03-01

    In this study, we give a thorough analysis of a general affine gravity with torsion. After a brief exposition of the affine gravities considered by Eddington and Schrödinger, we construct and analyze different affine gravities based on the determinants of the Ricci tensor, the torsion tensor, the Riemann tensor, and their combinations. In each case we reduce equations of motion to their simplest forms and give a detailed analysis of their solutions. Our analyses lead to the construction of the affine connection in terms of the curvature and torsion tensors. Our solutions of the dynamical equations show that the curvature tensors at different points are correlated via non-local, exponential rescaling factors determined by the torsion tensor.

  20. Lectin affinity electrophoresis.

    PubMed

    Kobayashi, Yuka

    2014-01-01

    An interaction or a binding event typically changes the electrophoretic properties of a molecule. Affinity electrophoresis methods detect changes in the electrophoretic pattern of molecules (mainly macromolecules) that occur as a result of biospecific interactions or complex formation. Lectin affinity electrophoresis is a very effective method for the detection and analysis of trace amounts of glycobiological substances. It is particularly useful for isolating and separating the glycoisomers of target molecules. Here, we describe a sensitive technique for the detection of glycoproteins separated by agarose gel-lectin affinity electrophoresis that uses antibody-affinity blotting. The technique is tested using α-fetoprotein with lectin (Lens culinaris agglutinin and Phaseolus vulgaris agglutinin)-agarose gels.

  1. Gas-phase nitronium ion affinities.

    PubMed Central

    Cacace, F; de Petris, G; Pepi, F; Angelelli, F

    1995-01-01

    Evaluation of nitronium ion-transfer equilibria, L1NO2+ + L2 = L2NO2+ + L1 (where L1 and L2 are ligands 1 and 2, respectively) by Fourier-transform ion cyclotron resonance mass spectrometry and application of the kinetic method, based on the metastable fragmentation of L1(NO2+)L2 nitronium ion-bound dimers led to a scale of relative gas-phase nitronium ion affinities. This scale, calibrated to a recent literature value for the NO2+ affinity of water, led for 18 ligands, including methanol, ammonia, representative ketones, nitriles, and nitroalkanes, to absolute NO2+ affinities, that fit a reasonably linear general correlation when plotted vs. the corresponding proton affinities (PAs). The slope of the plot depends to a certain extent on the specific nature of the ligands and, hence, the correlations between the NO2+ affinities, and the PAs of a given class of compounds display a better linearity than the general correlation and may afford a useful tool for predicting the NO2+ affinity of a molecule based on its PA. The NO2+ binding energies are considerably lower than the corresponding PAs and well below the binding energies of related polyatomic cations, such as NO+, a trend consistent with the available theoretical results on the structure and the stability of simple NO2+ complexes. The present study reports an example of extension of the kinetic method to dimers, such as L1(NO2+)L2, bound by polyatomic ions, which may considerably widen its scope. Finally, measurement of the NO2+ affinity of ammonia allowed evaluation of the otherwise inaccessible PA of the amino group of nitramide and, hence, direct experimental verification of previous theoretical estimates. PMID:11607578

  2. Imaging of uranium on rat brain sections using laser ablation inductively coupled plasma mass spectrometry: a new tool for the study of critical substructures affined to heavy metals in tissues.

    PubMed

    Becker, J Sabine; Dobrowolska, Justina; Zoriy, Miroslav; Matusch, Andreas

    2008-09-01

    The specific toxicity of trace metals and compounds largely depends on their bioavailability in different organs or compartments of the organism considered. Imaging mass spectrometry (IMS) using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) with a spatial resolution in the 100 microm range was developed and employed to study heavy metal distribution in brain tissues for toxicological screening. Rat brain post-mortem tissues were stained in an aqueous solution of either uranium or neodymium (metal concentration 100 microg g(-1)) for 3 h. The incubation of heavy metal in thin slices of brain tissue is followed by an imaging mass spectrometric LA-ICP-MS technique. Stained rat brain tissue (thickness 30 microm) were scanned with a focused laser beam (wavelength 266 nm, diameter of laser crater 100 microm and laser power density 3 x 10(9) W cm(-2)). The ion intensities of (235)U(+), (238)U(+), (145)Nd(+) and (146)Nd(+) were measured by LA-ICP-MS within the ablated area. For quantification purposes, matrix-matched laboratory standards were prepared by dosing each analyte to the pieces of homogenized brain tissue. Imaging LA-ICP-MS allows structures of interest to be identified and the relevant dose range to be estimated.

  3. A Method to Site-Specifically Identify and Quantitate Carbonyl End Products of Protein Oxidation Using Oxidation-Dependent Element Coded Affinity Tags (O-ECAT) and NanoLiquid Chromatography Fourier Transform Mass Spectrometry

    SciTech Connect

    Lee, S; Young, N L; Whetstone, P A; Cheal, S M; Benner, W H; Lebrilla, C B; Meares, C F

    2005-08-25

    Protein oxidation is linked to cellular stress, aging, and disease. Protein oxidations that result in reactive species are of particular interest, since these reactive oxidation products may react with other proteins or biomolecules in an unmediated and irreversible fashion, providing a potential marker for a variety of disease mechanisms. We have developed a novel system to identify and quantitate, relative to other states, the sites of oxidation on a given protein. A specially designed Oxidation-dependent carbonyl-specific Element-Coded Affinity Mass Tag (O-ECAT), AOD, ((S)-2-(4-(2-aminooxy)-acetamido)-benzyl)-1, 4, 7, 10-tetraazacyclododecane-N, N', N'', N'''-tetraacetic acid, is used to covalently tag the residues of a protein oxidized to aldehyde or keto end products. After proteolysis, the resulting AOD-tagged peptides are affinity purified, and analyzed by nanoLC-FTICR-MS, which provides high specificity in extracting co-eluting AOD mass pairs with a unique mass difference and affords relative quantitation based on isotopic ratios. Using this methodology, we have mapped the surface oxidation sites on a model protein, recombinant human serum albumin (rHSA) in its native form (as purchased) and after FeEDTA oxidation. A variety of modified amino acid residues including lysine, arginine, proline, histidine, threonine, aspartic and glutamic acids, were found to be oxidized to aldehyde and keto end products. The sensitivity of this methodology is shown by the number of peptides identified, twenty peptides on the native protein and twenty-nine after surface oxidation using FeEDTA and ascorbate. All identified peptides map to the surface of the HSA crystal structure validating this method for identifying oxidized amino acids on protein surfaces. In relative quantitation experiments between FeEDTA oxidation and native protein oxidation, identified sites showed different relative propensities towards oxidation independent of amino acid residue. We expect to extend

  4. Stability of flavin semiquinones in the gas phase: the electron affinity, proton affinity, and hydrogen atom affinity of lumiflavin.

    PubMed

    Zhang, Tianlan; Papson, Kaitlin; Ochran, Richard; Ridge, Douglas P

    2013-11-07

    Examination of electron transfer and proton transfer reactions of lumiflavin and proton transfer reactions of the lumiflavin radical anion by Fourier transform ion cyclotron resonance mass spectrometry is described. From the equilibrium constant determined for electron transfer between 1,4-naphthoquinone and lumiflavin the electron affinity of lumiflavin is deduced to be 1.86 ± 0.1 eV. Measurements of the rate constants and efficiencies for proton transfer reactions indicate that the proton affinity of the lumiflavin radical anion is between that of difluoroacetate (331.0 kcal/mol) and p-formyl-phenoxide (333.0 kcal/mol). Combining the electron affinity of lumiflavin with the proton affinity of the lumiflavin radical anion gives a lumiflavin hydrogen atom affinity of 59.7 ± 2.2 kcal/mol. The ΔG298 deduced from these results for adding an H atom to gas phase lumiflavin, 52.1 ± 2.2 kcal/mol, is in good agreement with ΔG298 for adding an H atom to aqueous lumiflavin from electrochemical measurements in the literature, 51.0 kcal/mol, and that from M06-L density functional calculations in the literature, 51.2 kcal/mol, suggesting little, if any, solvent effect on the H atom addition. The proton affinity of lumiflavin deduced from the equilibrium constant for the proton transfer reaction between lumiflavin and 2-picoline is 227.3 ± 2.0 kcal mol(-1). Density functional theory calculations on isomers of protonated lumiflavin provide a basis for assigning the most probable site of protonation as position 1 on the isoalloxazine ring and for estimating the ionization potentials of lumiflavin neutral radicals.

  5. Detection of protein-protein interactions using tandem affinity purification.

    PubMed

    Goodfellow, Ian; Bailey, Dalan

    2014-01-01

    Tandem affinity purification (TAP) is an invaluable technique for identifying interaction partners for an affinity tagged bait protein. The approach relies on the fusion of dual tags to the bait before separate rounds of affinity purification and precipitation. Frequently two specific elution steps are also performed to increase the specificity of the overall technique. In the method detailed here, the two tags used are protein G and a short streptavidin binding peptide; however, many variations can be employed. In our example the tags are separated by a cleavable tobacco etch virus protease target sequence, allowing for specific elution after the first round of affinity purification. Proteins isolated after the final elution step in this process are concentrated before being identified by mass spectrometry. The use of dual affinity tags and specific elution in this technique dramatically increases both the specificity and stringency of the pull-downs, ensuring a low level of background nonspecific interactions.

  6. Affine Sphere Relativity

    NASA Astrophysics Data System (ADS)

    Minguzzi, E.

    2017-03-01

    We investigate spacetimes whose light cones could be anisotropic. We prove the equivalence of the structures: (a) Lorentz-Finsler manifold for which the mean Cartan torsion vanishes, (b) Lorentz-Finsler manifold for which the indicatrix (observer space) at each point is a convex hyperbolic affine sphere centered on the zero section, and (c) pair given by a spacetime volume and a sharp convex cone distribution. The equivalence suggests to describe (affine sphere) spacetimes with this structure, so that no algebraic-metrical concept enters the definition. As a result, this work shows how the metric features of spacetime emerge from elementary concepts such as measure and order. Non-relativistic spacetimes are obtained replacing proper spheres with improper spheres, so the distinction does not call for group theoretical elements. In physical terms, in affine sphere spacetimes the light cone distribution and the spacetime measure determine the motion of massive and massless particles (hence the dispersion relation). Furthermore, it is shown that, more generally, for Lorentz-Finsler theories non-differentiable at the cone, the lightlike geodesics and the transport of the particle momentum over them are well defined, though the curve parametrization could be undefined. Causality theory is also well behaved. Several results for affine sphere spacetimes are presented. Some results in Finsler geometry, for instance in the characterization of Randers spaces, are also included.

  7. Kernel Affine Projection Algorithms

    NASA Astrophysics Data System (ADS)

    Liu, Weifeng; Príncipe, José C.

    2008-12-01

    The combination of the famed kernel trick and affine projection algorithms (APAs) yields powerful nonlinear extensions, named collectively here, KAPA. This paper is a follow-up study of the recently introduced kernel least-mean-square algorithm (KLMS). KAPA inherits the simplicity and online nature of KLMS while reducing its gradient noise, boosting performance. More interestingly, it provides a unifying model for several neural network techniques, including kernel least-mean-square algorithms, kernel adaline, sliding-window kernel recursive-least squares (KRLS), and regularization networks. Therefore, many insights can be gained into the basic relations among them and the tradeoff between computation complexity and performance. Several simulations illustrate its wide applicability.

  8. Affinity proteomics to study endogenous protein complexes: Pointers, pitfalls, preferences and perspectives

    PubMed Central

    LaCava, John; Molloy, Kelly R.; Taylor, Martin S.; Domanski, Michal; Chait, Brian T.; Rout, Michael P.

    2015-01-01

    Dissecting and studying cellular systems requires the ability to specifically isolate distinct proteins along with the co-assembled constituents of their associated complexes. Affinity capture techniques leverage high affinity, high specificity reagents to target and capture proteins of interest along with specifically associated proteins from cell extracts. Affinity capture coupled to mass spectrometry (MS)-based proteomic analyses has enabled the isolation and characterization of a wide range of endogenous protein complexes. Here, we outline effective procedures for the affinity capture of protein complexes, highlighting best practices and common pitfalls. PMID:25757543

  9. Ultrasensitive characterization of site-specific glycosylation of affinity-purified haptoglobin from lung cancer patient plasma using 10 μm i.d. porous layer open tubular liquid chromatography-linear ion trap collision-induced dissociation/electron transfer dissociation mass spectrometry.

    PubMed

    Wang, Dongdong; Hincapie, Marina; Rejtar, Tomas; Karger, Barry L

    2011-03-15

    Site-specific analysis of protein glycosylation is important for biochemical and clinical research efforts. Glycopeptide analysis using liquid chromatography-collision-induced dissociation/electron transfer dissociation mass spectrometry (LC-CID/ETD-MS) allows simultaneous characterization of the glycan structure and attached peptide site. However, due to the low ionization efficiency of glycopeptides during electrospray ionization, 200-500 fmol of sample per injection is needed for a single LC-MS run, which makes it challenging for the analysis of limited amounts of glycoprotein purified from biological matrixes. To improve the sensitivity of LC-MS analysis for glycopeptides, an ultranarrow porous layer open tubular (PLOT) LC column (2.5 m × 10 μm i.d.) was coupled to a linear ion trap (LTQ) collision-induced dissociation/electron transfer dissociation mass spectrometer to provide sensitive analysis of N-linked protein glycosylation heterogeneity. The potential of the developed method is demonstrated by the characterization of site-specific glycosylation using haptoglobin (Hpt) as a model protein. To limit the amount of haptoglobin to low picomole amounts of protein, we affinity purified it from 1 μL of pooled lung cancer patient plasma. A total of 26 glycoforms/glycan compositions on three Hpt tryptic glycopeptides were identified and quantified from 10 LC-MS runs with a consumption of 100 fmol of Hpt digest (13 ng of protein, 10 fmol per injection). Included in this analysis was the determination of the glycan occupancy level. At this sample consumption level, the high sensitivity of the PLOT LC-LTQ-CID/ETD-MS system allowed glycopeptide identification and structure determination, along with relative quantitation of glycans presented on the same peptide backbone, even for low abundant glycopeptides at the ∼100 amol level. The PLOT LC-MS system is shown to have sufficient sensitivity to allow characterization of site-specific protein glycosylation from trace

  10. Adjoint affine fusion and tadpoles

    NASA Astrophysics Data System (ADS)

    Urichuk, Andrew; Walton, Mark A.

    2016-06-01

    We study affine fusion with the adjoint representation. For simple Lie algebras, elementary and universal formulas determine the decomposition of a tensor product of an integrable highest-weight representation with the adjoint representation. Using the (refined) affine depth rule, we prove that equally striking results apply to adjoint affine fusion. For diagonal fusion, a coefficient equals the number of nonzero Dynkin labels of the relevant affine highest weight, minus 1. A nice lattice-polytope interpretation follows and allows the straightforward calculation of the genus-1 1-point adjoint Verlinde dimension, the adjoint affine fusion tadpole. Explicit formulas, (piecewise) polynomial in the level, are written for the adjoint tadpoles of all classical Lie algebras. We show that off-diagonal adjoint affine fusion is obtained from the corresponding tensor product by simply dropping non-dominant representations.

  11. Quantitative interaction proteomics using mass spectrometry.

    PubMed

    Wepf, Alexander; Glatter, Timo; Schmidt, Alexander; Aebersold, Ruedi; Gstaiger, Matthias

    2009-03-01

    We present a mass spectrometry-based strategy for the absolute quantification of protein complex components isolated through affinity purification. We quantified bait proteins via isotope-labeled reference peptides corresponding to an affinity tag sequence and prey proteins by label-free correlational quantification using the precursor ion signal intensities of proteotypic peptides generated in reciprocal purifications. We used this method to quantitatively analyze interaction stoichiometries in the human protein phosphatase 2A network.

  12. Mass spectrometry.

    NASA Technical Reports Server (NTRS)

    Burlingame, A. L.; Johanson, G. A.

    1972-01-01

    Review of the current state of mass spectrometry, indicating its unique importance for advanced scientific research. Mass spectrometry applications in computer techniques, gas chromatography, ion cyclotron resonance, molecular fragmentation and ionization, and isotope labeling are covered. Details are given on mass spectrometry applications in bio-organic chemistry and biomedical research. As the subjects of these applications are indicated alkaloids, carbohydrates, lipids, terpenes, quinones, nucleic acid components, peptides, antibiotics, and human and animal metabolisms. Particular attention is given to the mass spectra of organo-inorganic compounds, inorganic mass spectrometry, surface phenomena such as secondary ion and electron emission, and elemental and isotope analysis. Further topics include mass spectrometry in organic geochemistry, applications in geochronology and cosmochemistry, and organic mass spectrometry.

  13. Electron Affinity Calculations for Thioethers

    NASA Technical Reports Server (NTRS)

    Sulton, Deley L.; Boothe, Michael; Ball, David W.; Morales, Wilfredo

    1997-01-01

    Previous work indicated that polyphenyl thioethers possessed chemical properties, related to their electron affinities, which could allow them to function as vapor phase lubricants (VPL). Indeed, preliminary tribological tests revealed that the thioethers could function as vapor phase lubricants but not over a wide temperature and hertzian pressure range. Increasing the electron affinity of the thioethers may improve their VPL properties over this range. Adding a substituent group to the thioether will alter its electron affinity in many cases. Molecular orbital calculations were undertaken to determine the effect of five different substituent groups on the electron affinity of polyphenyl thioethers. It was found that the NO2, F, and I groups increased the thioethers electron affinity by the greatest amount. Future work will involve the addition of these groups to the thioethers followed by tribological testing to assess their VPL properties.

  14. Affinity-based target deconvolution of safranal

    PubMed Central

    2013-01-01

    Background and the purpose of the study Affinity-based target deconvolution is an emerging method for the identification of interactions between drugs/drug candidates and cellular proteins, and helps to predict potential activities and side effects of a given compound. In the present study, we hypothesized that a part of safranal pharmacological effects, one of the major constituent of Crocus sativus L., relies on its physical interaction with target proteins. Methods Affinity chromatography solid support was prepared by covalent attachment of safranal to agarose beads. After passing tissue lysate through the column, safranal-bound proteins were isolated and separated on SDS-PAGE or two-dimensional gel electrophoresis. Proteins were identified using MALDI-TOF/TOF mass spectrometry and Mascot software. Results and major conclusion Data showed that safranal physically binds to beta actin, cytochrome b-c1 complex sub-unit 1, trifunctional enzyme sub-unit beta and ATP synthase sub-unit alpha and beta. These interactions may explain part of safranal’s pharmacological effects. However, phenotypic and/or biological relevance of these interactions remains to be elucidated by future pharmacological studies. PMID:23514587

  15. The proton affinities of saturated and unsaturated heterocyclic molecules

    NASA Astrophysics Data System (ADS)

    Kabli, Samira; van Beelen, Eric S. E.; Ingemann, Steen; Henriksen, Lars; Hammerum, Steen

    2006-03-01

    The proton affinities derived from G3-calculations of 23 five-membered ring heteroaromatic molecules agree well with the experimentally determined values available in the literature. The calculated local proton affinities show that the principal site of protonation of the heteroaromatic compounds examined is an atom of the ring, carbon when there is only one heteroatom in the ring, and nitrogen where there are two or more heteroatoms. The experimental proton affinities of non-aromatic cyclic ethers, amines and thioethers are also in excellent agreement with the calculated values, with two exceptions (oxetane, N-methylazetidine). The literature proton affinities of the four simple cyclic ethers, oxetane, tetrahydrofuran, tetrahydropyran and oxepane were confirmed by Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass spectrometry, in order to examine the disagreement between the values predicted by extrapolation or additivity for tetrahydrofuran and tetrahydropyran and those determined by experiment and by calculation. The proton affinity differences between the pairs tetrahydropyran/1,4-dioxane, piperidine/morpholine and related compounds show that introduction of an additional oxygen atom in the ring considerably lowers the basicity.

  16. Raman Spectrometry.

    ERIC Educational Resources Information Center

    Gardiner, Derek J.

    1980-01-01

    Reviews mainly quantitative analytical applications in the field of Raman spectrometry. Includes references to other reviews, new and analytically untested techniques, and novel sampling and instrument designs. Cites 184 references. (CS)

  17. Boronate affinity materials for separation and molecular recognition: structure, properties and applications.

    PubMed

    Li, Daojin; Chen, Yang; Liu, Zhen

    2015-11-21

    Boronate affinity materials, as unique sorbents, have emerged as important media for the selective separation and molecular recognition of cis-diol-containing compounds. With the introduction of boronic acid functionality, boronate affinity materials exhibit several significant advantages, including broad-spectrum selectivity, reversible covalent binding, pH-controlled capture/release, fast association/desorption kinetics, and good compatibility with mass spectrometry. Because cis-diol-containing biomolecules, including nucleosides, saccharides, glycans, glycoproteins and so on, are the important targets in current research frontiers such as metabolomics, glycomics and proteomics, boronate affinity materials have gained rapid development and found increasing applications in the last decade. In this review, we critically survey recent advances in boronate affinity materials. We focus on fundamental considerations as well as important progress and new boronate affinity materials reported in the last decade. We particularly discuss on the effects of the structure of boronate ligands and supporting materials on the properties of boronate affinity materials, such as binding pH, affinity, selectivity, binding capacity, tolerance for interference and so on. A variety of promising applications, including affinity separation, proteomics, metabolomics, disease diagnostics and aptamer selection, are introduced with main emphasis on how boronate affinity materials can solve the issues in the applications and what merits boronate affinity materials can provide.

  18. Contractions of affine spherical varieties

    SciTech Connect

    Arzhantsev, I V

    1999-08-31

    The language of filtrations and contractions is used to describe the class of G-varieties obtainable as the total spaces of the construction of contraction applied to affine spherical varieties, which is well-known in invariant theory. These varieties are local models for arbitrary affine G-varieties of complexity 1 with a one-dimensional categorical quotient. As examples, reductive algebraic semigroups and three-dimensional SL{sub 2}-varieties are considered.

  19. Protein Complex Affinity Capture from Cryomilled Mammalian Cells.

    PubMed

    LaCava, John; Jiang, Hua; Rout, Michael P

    2016-12-09

    Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

  20. Protein Complex Affinity Capture from Cryomilled Mammalian Cells

    PubMed Central

    LaCava, John; Jiang, Hua; Rout, Michael P.

    2016-01-01

    Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets. PMID:28060343

  1. Mass spectrometry with accelerators.

    PubMed

    Litherland, A E; Zhao, X-L; Kieser, W E

    2011-01-01

    As one in a series of articles on Canadian contributions to mass spectrometry, this review begins with an outline of the history of accelerator mass spectrometry (AMS), noting roles played by researchers at three Canadian AMS laboratories. After a description of the unique features of AMS, three examples, (14)C, (10)Be, and (129)I are given to illustrate the methods. The capabilities of mass spectrometry have been extended by the addition of atomic isobar selection, molecular isobar attenuation, further ion acceleration, followed by ion detection and ion identification at essentially zero dark current or ion flux. This has been accomplished by exploiting the techniques and accelerators of atomic and nuclear physics. In 1939, the first principles of AMS were established using a cyclotron. In 1977 the selection of isobars in the ion source was established when it was shown that the (14)N(-) ion was very unstable, or extremely difficult to create, making a tandem electrostatic accelerator highly suitable for assisting the mass spectrometric measurement of the rare long-lived radioactive isotope (14)C in the environment. This observation, together with the large attenuation of the molecular isobars (13)CH(-) and (12)CH 2(-) during tandem acceleration and the observed very low background contamination from the ion source, was found to facilitate the mass spectrometry of (14)C to at least a level of (14)C/C ~ 6 × 10(-16), the equivalent of a radiocarbon age of 60,000 years. Tandem Accelerator Mass Spectrometry, or AMS, has now made possible the accurate radiocarbon dating of milligram-sized carbon samples by ion counting as well as dating and tracing with many other long-lived radioactive isotopes such as (10)Be, (26)Al, (36)Cl, and (129)I. The difficulty of obtaining large anion currents with low electron affinities and the difficulties of isobar separation, especially for the heavier mass ions, has prompted the use of molecular anions and the search for alternative

  2. Chemical binding affinity estimation using MSB

    NASA Astrophysics Data System (ADS)

    Weaver, John B.; Rauwerdink, Adam M.

    2011-03-01

    Binding affinity can be estimated in several ways in the laboratory but there is no viable way to estimate binding affinity in vivo without assumptions on the number of binding sites. Magnetic spectroscopy of nanoparticle Brownian motion, MSB, measures the rotational Brownian motion. The MSB signal is affected by nanoparticle binding affinity so it provides a mechanism to measure the chemical binding affinity. We present a possible mechanism to quantify the binding affinity and test that mechanism using viscous solutions.

  3. Affine Contractions on the Plane

    ERIC Educational Resources Information Center

    Celik, D.; Ozdemir, Y.; Ureyen, M.

    2007-01-01

    Contractions play a considerable role in the theory of fractals. However, it is not easy to find contractions which are not similitudes. In this study, it is shown by counter examples that an affine transformation of the plane carrying a given triangle onto another triangle may not be a contraction even if it contracts edges, heights or medians.…

  4. Affinity-aware checkpoint restart

    SciTech Connect

    Saini, Ajay; Rezaei, Arash; Mueller, Frank; Hargrove, Paul; Roman, Eric

    2014-12-08

    Current checkpointing techniques employed to overcome faults for HPC applications result in inferior application performance after restart from a checkpoint for a number of applications. This is due to a lack of page and core affinity awareness of the checkpoint/restart (C/R) mechanism, i.e., application tasks originally pinned to cores may be restarted on different cores, and in case of non-uniform memory architectures (NUMA), quite common today, memory pages associated with tasks on a NUMA node may be associated with a different NUMA node after restart. Here, this work contributes a novel design technique for C/R mechanisms to preserve task-to-core maps and NUMA node specific page affinities across restarts. Experimental results with BLCR, a C/R mechanism, enhanced with affinity awareness demonstrate significant performance benefits of 37%-73% for the NAS Parallel Benchmark codes and 6-12% for NAMD with negligible overheads instead of up to nearly four times longer an execution times without affinity-aware restarts on 16 cores.

  5. Affinity-aware checkpoint restart

    DOE PAGES

    Saini, Ajay; Rezaei, Arash; Mueller, Frank; ...

    2014-12-08

    Current checkpointing techniques employed to overcome faults for HPC applications result in inferior application performance after restart from a checkpoint for a number of applications. This is due to a lack of page and core affinity awareness of the checkpoint/restart (C/R) mechanism, i.e., application tasks originally pinned to cores may be restarted on different cores, and in case of non-uniform memory architectures (NUMA), quite common today, memory pages associated with tasks on a NUMA node may be associated with a different NUMA node after restart. Here, this work contributes a novel design technique for C/R mechanisms to preserve task-to-core mapsmore » and NUMA node specific page affinities across restarts. Experimental results with BLCR, a C/R mechanism, enhanced with affinity awareness demonstrate significant performance benefits of 37%-73% for the NAS Parallel Benchmark codes and 6-12% for NAMD with negligible overheads instead of up to nearly four times longer an execution times without affinity-aware restarts on 16 cores.« less

  6. ELECTRON AFFINITIES OF INORGANIC RADICALS.

    DTIC Science & Technology

    energy in the latter compound is 110 kcals/mole, distinctly higher than in ammonia. Cyanogen (CN)2 and hydrocyanic acid (HCN) yield values for the...ions very readily, and the electron affinity is 49 kcals/mole. A comparison with the results from thiocyanic acid (HNCS) indicates that the H-N bond

  7. Theoretical proton affinity and fluoride affinity of nerve agent VX.

    PubMed

    Bera, Narayan C; Maeda, Satoshi; Morokuma, Keiji; Viggiano, Al A

    2010-12-23

    Proton affinity and fluoride affinity of nerve agent VX at all of its possible sites were calculated at the RI-MP2/cc-pVTZ//B3LYP/6-31G* and RI-MP2/aug-cc-pVTZ//B3LYP/6-31+G* levels, respectively. The protonation leads to various unique structures, with H(+) attached to oxygen, nitrogen, and sulfur atoms; among which the nitrogen site possesses the highest proton affinity of -ΔE ∼ 251 kcal/mol, suggesting that this is likely to be the major product. In addition some H(2), CH(4) dissociation as well as destruction channels have been found, among which the CH(4) + [Et-O-P(═O)(Me)-S-(CH(2))(2)-N(+)(iPr)═CHMe] product and the destruction product forming Et-O-P(═O)(Me)-SMe + CH(2)═N(+)(iPr)(2) are only 9 kcal/mol less stable than the most stable N-protonated product. For fluoridization, the S-P destruction channel to give Et-O-P(═O)(Me)(F) + [S-(CH(2))(2)-N-(iPr)(2)](-) is energetically the most favorable, with a fluoride affinity of -ΔE ∼ 44 kcal. Various F(-) ion-molecule complexes are also found, with the one having F(-) interacting with two hydrogen atoms in different alkyl groups to be only 9 kcal/mol higher than the above destruction product. These results suggest VX behaves quite differently from surrogate systems.

  8. Dual-tagging system for the affinity purification of mammalian protein complexes

    SciTech Connect

    Giannone, Richard J; McDonald, W Hayes; Hurst, Gregory {Greg} B; Huang, Ying; Wu, Jun; Liu, Yie; Wang, Yisong

    2007-01-01

    Although affinity purification coupled with mass spectrometry (MS) provides a powerful tool to study protein-protein interactions, this strategy has encountered numerous difficulties when adapted to mammalian cells. Here we describe a Gateway{reg_sign}-compatible dual-tag affinity purification system that integrates regulatable expression, tetracysteine motifs, and various combinations of affinity tags to facilitate the cloning, detection, and purification of bait proteins and their interacting partners. Utilizing the human telomere binding protein TRF2 as a benchmark, we demonstrate bait protein recoveries upwards of approximately 16% from as little as 1-7 x 10{sup 7} cells and successfully identify known TRF2 interacting proteins, suggesting that our dual-tag affinity purification approach is a capable new tool for expanding the capacity to explore mammalian proteomic networks.

  9. Analysis of protein complexes using mass spectrometry.

    PubMed

    Gingras, Anne-Claude; Gstaiger, Matthias; Raught, Brian; Aebersold, Ruedi

    2007-08-01

    The versatile combination of affinity purification and mass spectrometry (AP-MS) has recently been applied to the detailed characterization of many protein complexes and large protein-interaction networks. The combination of AP-MS with other techniques, such as biochemical fractionation, intact mass measurement and chemical crosslinking, can help to decipher the supramolecular organization of protein complexes. AP-MS can also be combined with quantitative proteomics approaches to better understand the dynamics of protein-complex assembly.

  10. Mass spectrometry

    SciTech Connect

    Burlingame, A.L.; Baillie, T.A.; Derrick, P.J.

    1986-04-01

    It is the intention of the review to bring together in one source the direction of major developments in mass spectrometry and to illustrate these by citing key contributions from both fundamental and applied research. The Review is intended to provide the reader with a sense of the main currents, their breadth and depth, and probable future directions. It is also intended to provide the reader with a glimpse of the diverse discoveries and results that underpin the eventual development of new methods and instruments - the keys to obtaining new insights in all the physical, chemical, and biological sciences which depend on mass spectrometry at various levels of sophistication. Focal points for future interdisciplinary synergism might be selective quantitative derivatization of large peptides, which would convey properties that direct fragmentation providing specific sequence information, or optimization of LCMS for biooligomer sequencing and mixture analysis, or the perfect way to control or enhance the internal energy of ions of any size, or many others. 1669 references.

  11. Production and Characterization of Desmalonichrome Relative Binding Affinity for Uranyl Ions in Relation to Other Siderophores

    SciTech Connect

    Mo, Kai-For; Dai, Ziyu; Wunschel, David S.

    2016-06-24

    Siderophores are Fe binding secondary metabolites that have been investigated for their uranium binding properties. Much of the previous work has focused on characterizing hydroxamate types of siderophores, such as desferrioxamine B, for their uranyl binding affinity. Carboxylate forms of these metabolites hold potential to be more efficient chelators of uranyl, yet they have not been widely studied and are more difficult to obtain. Desmalonichrome is a carboxylate siderophore which is not commercially available and so was obtained from the ascomycete fungus Fusarium oxysporum cultivated under Fe depleted conditions. The relative affinity for uranyl binding of desmalonichrome was investigated using a competitive analysis of binding affinities between uranyl acetate and different concentrations of iron(III) chloride using electrospray ionization mass spectrometry (ESI-MS). In addition to desmalonichrome, three other siderophores, including two hydroxamates (desferrioxamine B and desferrichrome) and one carboxylate (desferrichrome A) were studied to understand their relative affinities for the uranyl ion at two pH values. The binding affinities of hydroxymate siderophores to uranyl ion were found to decrease to a greater degree at lower pH as the concentration of Fe (III) ion increases. On the other hand, lowering pH has little impact on the binding affinities between carboxylate siderophores and uranyl ion. Desmalonichrome was shown to have the greatest relative affinity for uranyl at any pH and Fe(III) concentration. These results suggest that acidic functional groups in the ligands are critical for strong chelation with uranium at lower pH.

  12. Application of mass spectrometry in proteomics.

    PubMed

    Guerrera, Ida Chiara; Kleiner, Oliver

    2005-01-01

    Mass spectrometry has arguably become the core technology in proteomics. The application of mass spectrometry based techniques for the qualitative and quantitative analysis of global proteome samples derived from complex mixtures has had a big impact in the understanding of cellular function. Here, we give a brief introduction to principles of mass spectrometry and instrumentation currently used in proteomics experiments. In addition, recent developments in the application of mass spectrometry in proteomics are summarised. Strategies allowing high-throughput identification of proteins from highly complex mixtures include accurate mass measurement of peptides derived from total proteome digests and multidimensional peptide separations coupled with mass spectrometry. Mass spectrometric analysis of intact proteins permits the characterisation of protein isoforms. Recent developments in stable isotope labelling techniques and chemical tagging allow the mass spectrometry based differential display and quantitation of proteins, and newly established affinity procedures enable the targeted characterisation of post-translationally modified proteins. Finally, advances in mass spectrometric imaging allow the gathering of specific information on the local molecular composition, relative abundance and spatial distribution of peptides and proteins in thin tissue sections.

  13. Biotin-Streptavidin Affinity Purification of RNA-Protein Complexes Assembled In Vitro.

    PubMed

    Hou, Shuai; Shi, Lei; Lei, Haixin

    2016-01-01

    RNA-protein complexes are essential for the function of different RNAs, yet purification of specific RNA-protein complexes can be complicated and is a major obstacle in understanding the mechanism of regulatory RNAs. Here we present a protocol to purify RNA-protein complexes assembled in vitro based on biotin-streptavidin affinity. In vitro transcribed RNA is labeled with (32)P and biotin, ribonucleoprotein particles or RNPs are assembled by incubation of RNA in nuclear extract and fractionated using gel filtration, and RNP fractions are pooled for biotin-streptavidin affinity purification. The amount of RNA-protein complexes purified following this protocol is sufficient for mass spectrometry.

  14. Antisymmetric tensor generalizations of affine vector fields

    PubMed Central

    Morisawa, Yoshiyuki; Tomoda, Kentaro

    2016-01-01

    Tensor generalizations of affine vector fields called symmetric and antisymmetric affine tensor fields are discussed as symmetry of spacetimes. We review the properties of the symmetric ones, which have been studied in earlier works, and investigate the properties of the antisymmetric ones, which are the main theme in this paper. It is shown that antisymmetric affine tensor fields are closely related to one-lower-rank antisymmetric tensor fields which are parallelly transported along geodesics. It is also shown that the number of linear independent rank-p antisymmetric affine tensor fields in n-dimensions is bounded by (n + 1)!/p!(n − p)!. We also derive the integrability conditions for antisymmetric affine tensor fields. Using the integrability conditions, we discuss the existence of antisymmetric affine tensor fields on various spacetimes. PMID:26858463

  15. Conformal field theory on affine Lie groups

    SciTech Connect

    Clubok, Kenneth Sherman

    1996-04-01

    Working directly on affine Lie groups, we construct several new formulations of the WZW model, the gauged WZW model, and the generic affine-Virasoro action. In one formulation each of these conformal field theories (CFTs) is expressed as a one-dimensional mechanical system whose variables are coordinates on the affine Lie group. When written in terms of the affine group element, this formulation exhibits a two-dimensional WZW term. In another formulation each CFT is written as a two-dimensional field theory, with a three- dimensional WZW term, whose fields are coordinates on the affine group. On the basis of these equivalent formulations, we develop a translation dictionary in which the new formulations on the affine Lie group are understood as mode formulations of the conventional formulations on the Lie group. Using this dictionary, we also express each CFT as a three-dimensional field theory on the Lie group with a four-dimensional WZW term. 36 refs.

  16. A Novel Vertex Affinity for Community Detection

    SciTech Connect

    Yoo, Andy; Sanders, Geoffrey; Henson, Van; Vassilevski, Panayot

    2015-10-05

    We propose a novel vertex affinity measure in this paper. The new vertex affinity quantifies the proximity between two vertices in terms of their clustering strength and is ideal for such graph analytics applications as community detection. We also developed a framework that combines simple graph searches and resistance circuit formulas to compute the vertex affinity efficiently. We study the properties of the new affinity measure empirically in comparison to those of other popular vertex proximity metrics. Our results show that the existing metrics are ill-suited for community detection due to their lack of fundamental properties that are essential for correctly capturing inter- and intra-cluster vertex proximity.

  17. Structural determinants of sigma receptor affinity

    SciTech Connect

    Largent, B.L.; Wikstroem, H.G.; Gundlach, A.L.; Snyder, S.H.

    1987-12-01

    The structural determinants of sigma receptor affinity have been evaluated by examining a wide range of compounds related to opioids, neuroleptics, and phenylpiperidine dopaminergic structures for affinity at sigma receptor-binding sites labeled with (+)-(/sup 3/H)3-PPP. Among opioid compounds, requirements for sigma receptor affinity differ strikingly from the determinants of affinity for conventional opiate receptors. Sigma sites display reverse stereoselectivity to classical opiate receptors. Multi-ringed opiate-related compounds such as morphine and naloxone have negligible affinity for sigma sites, with the highest sigma receptor affinity apparent for benzomorphans which lack the C ring of opioids. Highest affinity among opioids and other compounds occurs with more lipophilic N-substituents. This feature is particularly striking among the 3-PPP derivatives as well as the opioids. The butyrophenone haloperidol is the most potent drug at sigma receptors we have detected. Among the series of butyrophenones, receptor affinity is primarily associated with the 4-phenylpiperidine moiety. Conformational calculations for various compounds indicate a fairly wide range of tolerance for distances between the aromatic ring and the amine nitrogen, which may account for the potency at sigma receptors of structures of considerable diversity. Among the wide range of structures that bind to sigma receptor-binding sites, the common pharmacophore associated with high receptor affinity is a phenylpiperidine with a lipophilic N-substituent.

  18. Structure of classical affine and classical affine fractional W-algebras

    SciTech Connect

    Suh, Uhi Rinn

    2015-01-15

    We introduce a classical BRST complex (See Definition 3.2.) and show that one can construct a classical affine W-algebra via the complex. This definition clarifies that classical affine W-algebras can be considered as quasi-classical limits of quantum affine W-algebras. We also give a definition of a classical affine fractional W-algebra as a Poisson vertex algebra. As in the classical affine case, a classical affine fractional W-algebra has two compatible λ-brackets and is isomorphic to an algebra of differential polynomials as a differential algebra. When a classical affine fractional W-algebra is associated to a minimal nilpotent, we describe explicit forms of free generators and compute λ-brackets between them. Provided some assumptions on a classical affine fractional W-algebra, we find an infinite sequence of integrable systems related to the algebra, using the generalized Drinfel’d and Sokolov reduction.

  19. Improving image segmentation by learning region affinities

    SciTech Connect

    Prasad, Lakshman; Yang, Xingwei; Latecki, Longin J

    2010-11-03

    We utilize the context information of other regions in hierarchical image segmentation to learn new regions affinities. It is well known that a single choice of quantization of an image space is highly unlikely to be a common optimal quantization level for all categories. Each level of quantization has its own benefits. Therefore, we utilize the hierarchical information among different quantizations as well as spatial proximity of their regions. The proposed affinity learning takes into account higher order relations among image regions, both local and long range relations, making it robust to instabilities and errors of the original, pairwise region affinities. Once the learnt affinities are obtained, we use a standard image segmentation algorithm to get the final segmentation. Moreover, the learnt affinities can be naturally unutilized in interactive segmentation. Experimental results on Berkeley Segmentation Dataset and MSRC Object Recognition Dataset are comparable and in some aspects better than the state-of-art methods.

  20. Innate immunity probed by lipopolysaccharides affinity strategy and proteomics.

    PubMed

    Giangrande, Chiara; Colarusso, Lucia; Lanzetta, Rosa; Molinaro, Antonio; Pucci, Piero; Amoresano, Angela

    2013-01-01

    Lipopolysaccharides (LPSs) are ubiquitous and vital components of the cell surface of Gram-negative bacteria that have been shown to play a relevant role in the induction of the immune-system response. In animal and plant cells, innate immune defenses toward microorganisms are triggered by the perception of pathogen associated molecular patterns. These are conserved and generally indispensable microbial structures such as LPSs that are fundamental in the Gram-negative immunity recognition. This paper reports the development of an integrated strategy based on lipopolysaccharide affinity methodology that represents a new starting point to elucidate the molecular mechanisms elicited by bacterial LPS and involved in the different steps of innate immunity response. Biotin-tagged LPS was immobilized on streptavidin column and used as a bait in an affinity capture procedure to identify protein partners from human serum specifically interacting with this effector. The complex proteins/lipopolysaccharide was isolated and the protein partners were fractionated by gel electrophoresis and identified by mass spectrometry. This procedure proved to be very effective in specifically binding proteins functionally correlated with the biological role of LPS. Proteins specifically bound to LPS essentially gathered within two functional groups, regulation of the complement system (factor H, C4b, C4BP, and alpha 2 macroglobulin) and inhibition of LPS-induced inflammation (HRG and Apolipoproteins). The reported strategy might have important applications in the elucidation of biological mechanisms involved in the LPSs-mediated molecular recognition and anti-infection responses.

  1. Reversible cyclic peptide libraries for the discovery of affinity ligands.

    PubMed

    Menegatti, Stefano; Ward, Kevin Lawrence; Naik, Amith Dattatray; Kish, William Stanley; Blackburn, Robert Kevin; Carbonell, Ruben Guillermo

    2013-10-01

    A novel strategy is presented for the identification of cyclic peptide ligands from combinatorial libraries of reversible cyclic depsipeptides. A method for the solid-phase synthesis of individual cyclic depsipeptides and combinatorial libraries of these compounds is proposed, which employs lactic acid (Lact) and the dipeptide ester (Nα-Ac)-Ser(Ala)- as linkers for dilactonization. Upon alkaline treatment of the beads selected by screening a model library, the cyclic depsipeptides are linearized and released from the solid support to the liquid phase, to be sequenced via single-step tandem mass spectrometry (MS/MS). The protocol presented for library synthesis provides for wide structural diversity. Two model sequences, VVWVVK and AAWAAR, were chosen to present different structural examples for depsipeptide libraries and demonstrate the process of sequence determination by mass spectrometry. Further, a case study using the IgG binding cyclic depsipeptide cyclo[(Nα-Ac)-S(A)-RWHYFK-Lact-E] is presented to demonstrate the process of library screening and sequence determination on the selected beads. Finally, a method is shown for synthesis of the irreversible cyclic peptide corresponding to the proposed depsipeptide structure, to make the ligand stable to the aqueous acid and alkaline conditions encountered in affinity chromatographic applications. The cyclic peptide ligand was synthesized on a poly(methacrylate) resin and used for chromatographic binding of the target IgG.

  2. The Cutting Edge of Affinity Electrophoresis Technology

    PubMed Central

    Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru

    2015-01-01

    Affinity electrophoresis is an important technique that is widely used to separate and analyze biomolecules in the fields of biology and medicine. Both quantitative and qualitative information can be gained through affinity electrophoresis. Affinity electrophoresis can be applied through a variety of strategies, such as mobility shift electrophoresis, charge shift electrophoresis or capillary affinity electrophoresis. These strategies are based on changes in the electrophoretic patterns of biological macromolecules that result from interactions or complex-formation processes that induce changes in the size or total charge of the molecules. Nucleic acid fragments can be characterized through their affinity to other molecules, for example transcriptional factor proteins. Hydrophobic membrane proteins can be identified by means of a shift in the mobility induced by a charged detergent. The various strategies have also been used in the estimation of association/disassociation constants. Some of these strategies have similarities to affinity chromatography, in that they use a probe or ligand immobilized on a supported matrix for electrophoresis. Such methods have recently contributed to profiling of major posttranslational modifications of proteins, such as glycosylation or phosphorylation. Here, we describe advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years. PMID:28248262

  3. Biomarker Candidates of Chlamydophila pneumoniae Proteins and Protein Fragments Identified by Affinity-Proteomics Using FTICR-MS and LC-MS/MS

    NASA Astrophysics Data System (ADS)

    Susnea, Iuliana; Bunk, Sebastian; Wendel, Albrecht; Hermann, Corinna; Przybylski, Michael

    2011-04-01

    We report here an affinity-proteomics approach that combines 2D-gel electrophoresis and immunoblotting with high performance mass spectrometry to the identification of both full length protein antigens and antigenic fragments of Chlamydophila pneumoniae (C. pneumoniae). The present affinity-mass spectrometry approach effectively utilized high resolution FTICR mass spectrometry and LC-tandem-MS for protein identification, and enabled the identification of several new highly antigenic C. pneumoniae proteins that were not hitherto reported or previously detected only in other Chlamydia species, such as Chlamydia trachomatis. Moreover, high resolution affinity-MS provided the identification of several neo-antigenic protein fragments containing N- and C-terminal, and central domains such as fragments of the membrane protein Pmp21 and the secreted chlamydial proteasome-like factor (Cpaf), representing specific biomarker candidates.

  4. Visualizing Antibody Affinity Maturation in Germinal Centers

    PubMed Central

    Tas, Jeroen M.J.; Mesin, Luka; Pasqual, Giulia; Targ, Sasha; Jacobsen, Johanne T.; Mano, Yasuko M.; Chen, Casie S.; Weill, Jean-Claude; Reynaud, Claude-Agnès; Browne, Edward P.; Meyer-Hermann, Michael; Victora, Gabriel D.

    2016-01-01

    Antibodies somatically mutate to attain high affinity in germinal centers (GCs). There, competition between B cell clones and among somatic mutants of each clone drives an increase in average affinity across the population. The extent to which higher-affinity cells eliminating competitors restricts clonal diversity is unknown. By combining multiphoton microscopy and sequencing, we show that tens to hundreds of distinct B cell clones seed each GC, and that GCs lose clonal diversity at widely disparate rates. Furthermore, efficient affinity maturation can occur in the absence of homogenizing selection, ensuring that many clones can mature in parallel within the same GC. Our findings have implications for development of vaccines in which antibodies with non-immunodominant specificities must be elicited, as is the case for HIV-1 and influenza. PMID:26912368

  5. PRINCIPLES OF AFFINITY-BASED BIOSENSORS

    EPA Science Inventory

    Despite the amount of resources that have been invested by national and international academic, government, and commercial sectors to develop affinity-based biosensor products, little obvious success has been realized through commercialization of these devices for specific applic...

  6. Protein purification using PDZ affinity chromatography.

    PubMed

    Walkup, Ward G; Kennedy, Mary B

    2015-04-01

    PDZ domains function in nature as protein-binding domains within scaffold and membrane-associated proteins. They comprise approximately 90 residues and undergo specific, high-affinity interactions with complementary C-terminal peptide sequences, other PDZ domains, and/or phospholipids. We have previously shown that the specific, strong interactions of PDZ domains with their ligands make them well suited for use in affinity chromatography. This unit provides protocols for the PDZ affinity chromatography procedure that are applicable for the purification of proteins that contain PDZ domains or PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We detail the preparation of affinity resins composed of PDZ domains or PDZ domain peptide ligands coupled to solid supports. These resins can be used to purify proteins containing endogenous or genetically introduced PDZ domains or ligands, eluting the proteins with free PDZ domain peptide ligands.

  7. Affinity Electrophoresis Using Ligands Attached To Polymers

    NASA Technical Reports Server (NTRS)

    Van Alstine, James M.; Snyder, Robert S.; Harris, J. M.; Brooks, D. E.

    1990-01-01

    In new technique, reduction of electrophoretic mobilities by addition of polyethylene glycol to ligands increases electrophoretic separabilities. In immuno-affinity electrophoresis, modification of ligands extends specificity of electrophoretic separation to particles having surface electric-charge structures otherwise making them electrophoretically inseparable. Modification of antibodies by polyethylene glycol greatly reduces ability to aggregate while enhancing ability to affect electrophoretic mobilities of cells. In hydrophobic-affinity electrophoresis, addition of polyethylene glycol reduces tendency toward aggregation of cells or macromolecules.

  8. A novel multiple affinity purification tag and its use in identification of proteins associated with a cyclin-CDK complex.

    PubMed

    Honey, S; Schneider, B L; Schieltz, D M; Yates, J R; Futcher, B

    2001-02-15

    A novel multiple affinity purification (MAFT) or tandem affinity purification (TAP) tag has been constructed. It consists of the calmodulin binding peptide, six histidine residues, and three copies of the hemagglutinin epitope. This 'CHH' MAFT tag allows two or three consecutive purification steps, giving high purity. Active Clb2-Cdc28 kinase complex was purified from yeast cells after inserting the CHH tag into Clb2. Associated proteins were identified using mass spectrometry. These included the known associated proteins Cdc28, Sic1 and Cks1. Several other proteins were found including the 70 kDa chaperone, Ssa1.

  9. Identification of proteins interacting with ammodytoxins in Vipera ammodytes ammodytes venom by immuno-affinity chromatography.

    PubMed

    Brgles, Marija; Kurtović, Tihana; Kovačič, Lidija; Križaj, Igor; Barut, Miloš; Lang Balija, Maja; Allmaier, Günter; Marchetti-Deschmann, Martina; Halassy, Beata

    2014-01-01

    In order to perform their function, proteins frequently interact with other proteins. Various methods are used to reveal protein interacting partners, and affinity chromatography is one of them. Snake venom is composed mostly of proteins, and various protein complexes in the venom have been found to exhibit higher toxicity levels than respective components separately. Complexes can modulate envenomation activity of a venom and/or potentiate its effect. Our previous data indicate that the most toxic components of the Vipera ammodytes ammodytes (Vaa) venom isolated so far-ammodytoxins (Atxs)-are contributing to the venom's toxicity only moderately; therefore, we aimed to explore whether they have some interacting partner(s) potentiating toxicity. For screening of possible interactions, immuno-affinity chromatography combined with identification by mass spectrometry was used. Various chemistries (epoxy, carbonyldiimidazole, ethylenediamine) as well as protein G functionality were used to immobilize antibodies on monolith support, a Convective Interaction Media disk. Monoliths have been demonstrated to better suit the separation of large biomolecules. Using such approach, several proteins were indicated as potential Atx-binding proteins. Among these, the interaction of Atxs with a Kunitz-type inhibitor was confirmed by far-Western dot-blot and surface plasmon resonance measurement. It can be concluded that affinity chromatography on monolithic columns combined with mass spectrometry identification is a successful approach for screening of protein interactions and it resulted with detection of the interaction of Atx with Kunitz-type inhibitor in Vaa venom for the first time.

  10. Proton Affinity Calculations with High Level Methods.

    PubMed

    Kolboe, Stein

    2014-08-12

    Proton affinities, stretching from small reference compounds, up to the methylbenzenes and naphthalene and anthracene, have been calculated with high accuracy computational methods, viz. W1BD, G4, G3B3, CBS-QB3, and M06-2X. Computed and the currently accepted reference proton affinities are generally in excellent accord, but there are deviations. The literature value for propene appears to be 6-7 kJ/mol too high. Reported proton affinities for the methylbenzenes seem 4-5 kJ/mol too high. G4 and G3 computations generally give results in good accord with the high level W1BD. Proton affinity values computed with the CBS-QB3 scheme are too low, and the error increases with increasing molecule size, reaching nearly 10 kJ/mol for the xylenes. The functional M06-2X fails markedly for some of the small reference compounds, in particular, for CO and ketene, but calculates methylbenzene proton affinities with high accuracy.

  11. Classification of neocortical interneurons using affinity propagation.

    PubMed

    Santana, Roberto; McGarry, Laura M; Bielza, Concha; Larrañaga, Pedro; Yuste, Rafael

    2013-01-01

    In spite of over a century of research on cortical circuits, it is still unknown how many classes of cortical neurons exist. In fact, neuronal classification is a difficult problem because it is unclear how to designate a neuronal cell class and what are the best characteristics to define them. Recently, unsupervised classifications using cluster analysis based on morphological, physiological, or molecular characteristics, have provided quantitative and unbiased identification of distinct neuronal subtypes, when applied to selected datasets. However, better and more robust classification methods are needed for increasingly complex and larger datasets. Here, we explored the use of affinity propagation, a recently developed unsupervised classification algorithm imported from machine learning, which gives a representative example or exemplar for each cluster. As a case study, we applied affinity propagation to a test dataset of 337 interneurons belonging to four subtypes, previously identified based on morphological and physiological characteristics. We found that affinity propagation correctly classified most of the neurons in a blind, non-supervised manner. Affinity propagation outperformed Ward's method, a current standard clustering approach, in classifying the neurons into 4 subtypes. Affinity propagation could therefore be used in future studies to validly classify neurons, as a first step to help reverse engineer neural circuits.

  12. Classification of neocortical interneurons using affinity propagation

    PubMed Central

    Santana, Roberto; McGarry, Laura M.; Bielza, Concha; Larrañaga, Pedro; Yuste, Rafael

    2013-01-01

    In spite of over a century of research on cortical circuits, it is still unknown how many classes of cortical neurons exist. In fact, neuronal classification is a difficult problem because it is unclear how to designate a neuronal cell class and what are the best characteristics to define them. Recently, unsupervised classifications using cluster analysis based on morphological, physiological, or molecular characteristics, have provided quantitative and unbiased identification of distinct neuronal subtypes, when applied to selected datasets. However, better and more robust classification methods are needed for increasingly complex and larger datasets. Here, we explored the use of affinity propagation, a recently developed unsupervised classification algorithm imported from machine learning, which gives a representative example or exemplar for each cluster. As a case study, we applied affinity propagation to a test dataset of 337 interneurons belonging to four subtypes, previously identified based on morphological and physiological characteristics. We found that affinity propagation correctly classified most of the neurons in a blind, non-supervised manner. Affinity propagation outperformed Ward's method, a current standard clustering approach, in classifying the neurons into 4 subtypes. Affinity propagation could therefore be used in future studies to validly classify neurons, as a first step to help reverse engineer neural circuits. PMID:24348339

  13. Identity, Affinity, Reality: Making the Case for Affinity Groups in Elementary School

    ERIC Educational Resources Information Center

    Parsons, Julie; Ridley, Kimberly

    2012-01-01

    Affinity groups are places where students build connections and process "ouch" moments from their classes. Children talk about the isolation they sometimes feel. The relationships students gain through race-based affinity groups enable them to feel less alone with their emotions and help them build a stronger sense of self. At the same…

  14. Stepparents' Affinity-Seeking and Affinity-Maintaining Strategies with Stepchildren.

    ERIC Educational Resources Information Center

    Ganong, Lawrence; Coleman, Marilyn; Fine, Mark; Martin, Patricia

    1999-01-01

    Examines the strategies that stepparents use to develop and maintain affinity with stepchildren and the effects that these strategies have on the development of stepparent-stepchildren relationships. Thirty-one affinity-seeking strategies are identified. Results show that dyadic activities worked best, but it is important that stepchildren…

  15. Affine coherent states and Toeplitz operators

    NASA Astrophysics Data System (ADS)

    Hutníková, Mária; Hutník, Ondrej

    2012-06-01

    We study a parameterized family of Toeplitz operators in the context of affine coherent states based on the Calderón reproducing formula (= resolution of unity on L_2( {R})) and the specific admissible wavelets (= affine coherent states in L_2( {R})) related to Laguerre functions. Symbols of such Calderón-Toeplitz operators as individual coordinates of the affine group (= upper half-plane with the hyperbolic geometry) are considered. In this case, a certain class of pseudo-differential operators, their properties and their operator algebras are investigated. As a result of this study, the Fredholm symbol algebras of the Calderón-Toeplitz operator algebras for these particular cases of symbols are described. This article is part of a special issue of Journal of Physics A: Mathematical and Theoretical devoted to ‘Coherent states: mathematical and physical aspects’.

  16. Non-affine elasticity in jammed systems

    NASA Astrophysics Data System (ADS)

    Maloney, Craig

    2006-03-01

    Symmetry dictates that perfect crystals should deform homogeneously, or affinely, under external load, and computing the elastic moduli from the underlying interaction potential is then straightforward. For disordered materials no such simple procedure exists, and recent numerical works have demonstrated that non-affine corrections can dramatically reduce the naive expectation for the shear modulus in a broad class of disordered systems and may control rigidity loss in the zero pressure limit in purely repulsive systems, i.e. the unjamming transition (c.f. [O'Hern et. al. PRE 68, 011306 (2003)]). We present numerical results and an analytical framework for the study of these non-affine corrections to the elastic response of disordered packings.

  17. Biomimetic affinity ligands for protein purification.

    PubMed

    Sousa, Isabel T; Taipa, M Angela

    2014-01-01

    The development of sophisticated molecular modeling software and new bioinformatic tools, as well as the emergence of data banks containing detailed information about a huge number of proteins, enabled the de novo intelligent design of synthetic affinity ligands. Such synthetic compounds can be tailored to mimic natural biological recognition motifs or to interact with key surface-exposed residues on target proteins and are designated as "biomimetic ligands." A well-established methodology for generating biomimetic or synthetic affinity ligands integrates rational design with combinatorial solid-phase synthesis and screening, using the triazine scaffold and analogues of amino acids side chains to create molecular diversity.Triazine-based synthetic ligands are nontoxic, low-cost, highly stable compounds that can replace advantageously natural biological ligands in the purification of proteins by affinity-based methodologies.

  18. Use of Affinity Diagrams as Instructional Tools in Inclusive Classrooms.

    ERIC Educational Resources Information Center

    Haselden, Polly G.

    2003-01-01

    This article describes how the affinity diagram, a tool for gathering information and organizing it into natural groupings, can be used in inclusive classrooms. It discusses how students can be taught to use an affinity diagram, how affinity diagrams can be used to reflect many voices, and how affinity diagrams can be used to plan class projects.…

  19. Cation affinity numbers of Lewis bases.

    PubMed

    Lindner, Christoph; Tandon, Raman; Maryasin, Boris; Larionov, Evgeny; Zipse, Hendrik

    2012-01-01

    Using selected theoretical methods the affinity of a large range of Lewis bases towards model cations has been quantified. The range of model cations includes the methyl cation as the smallest carbon-centered electrophile, the benzhydryl and trityl cations as models for electrophilic substrates encountered in Lewis base-catalyzed synthetic procedures, and the acetyl cation as a substrate model for acyl-transfer reactions. Affinities towards these cationic electrophiles are complemented by data for Lewis-base addition to Michael acceptors as prototypical neutral electrophiles.

  20. New unitary affine-Virasoro constructions

    SciTech Connect

    Halpern, M.B.; Kiritsis, E.; Obers, N.A.; Poratti, M. ); Yamron, J.P. )

    1990-06-20

    This paper reports on a quasi-systematic investigation of the Virasoro master equation. The space of all affine-Virasoro constructions is organized by K-conjugation into affine-Virasoro nests, and an estimate of the dimension of the space shows that most solutions await discovery. With consistent ansatze for the master equation, large classes of new unitary nests are constructed, including quadratic deformation nests with continuous conformal weights, and unitary irrational central charge nests, which may dominate unitary rational central charge on compact g.

  1. On the electron affinity of B2

    SciTech Connect

    Glezakou, Vanda A.; Taylor, Peter

    2009-02-02

    We present the results of high-level ab initio calculations on the electron affinity of B2. Our new best estimate of 1.93±0.03 eV is in agreement with previous calculations as well as the sole existing experimental estimate of 1.8 eV, as derived from quantities with an uncertainty of 0.4 eV. The electron affinity of atomic boron, which is much smaller, is also calculated for comparison, and again found to be in good agreement with experiment. Pacific Northwest National Laboratory is operated by Battelle for the US Department of Energy.

  2. Negative Electron Affinity Mechanism for Diamond Surfaces

    NASA Technical Reports Server (NTRS)

    Krainsky, I. L.; Asnin, V. M.

    1998-01-01

    The energy distribution of the secondary electrons for chemical vacuum deposited diamond films with Negative Electron Affinity (NEA) was investigated. It was found that while for completely hydrogenated diamond surfaces the negative electron affinity peak in the energy spectrum of the secondary electrons is present for any energy of the primary electrons, for partially hydrogenated diamond surfaces there is a critical energy above which the peak is present in the spectrum. This critical energy increases sharply when hydrogen coverage of the diamond surface diminishes. This effect was explained by the change of the NEA from the true type for the completely hydrogenated surface to the effective type for the partially hydrogenated surfaces.

  3. Evidence of multi-affinity in the Japanese stock market

    NASA Astrophysics Data System (ADS)

    Katsuragi, Hiroaki

    2000-04-01

    Fluctuations of the Japanese stock market (Tokyo Stock Price Index: TOPIX) are analyzed using a multi-affine analysis method. In the research to date, only some simulated self-affine models have shown multi-affinity. In most experiments using observations of self-affine fractal profiles, multi-affinity has not been found. However, we find evidence of multi-affinity in fluctuations of the Japanese stock market (TOPIX). The qth-order Hurst exponent Hq varies with changes in q. This multi-affinity indicates that there are plural mechanisms that affect the same time scale as stock market price fluctuation dynamics.

  4. Preparation and affinity identification of glutamic acid-urea small molecule analogs in prostate cancer

    PubMed Central

    Zhang, Zhiwei; Zhu, Zheng; Yang, Deyong; Fan, Weiwei; Wang, Jianbo; Li, Xiancheng; Chen, Xiaochi; Wang, Qifeng; Song, Xishuang

    2016-01-01

    In recent years, study concerning activity inhibitors of prostate-specific membrane antigen (PSMA) has been concentrated on the glutamic urea (Glu-urea-R) small molecule and its analogs. The present study aimed to synthesize 4 analogs of Glu-urea-R and identify the affinities of these compounds to PSMA. The compounds were synthesized from raw materials, and the experimental procedures of the present study were in accordance with standard techniques under anhydrous and anaerobic conditions. Glu-urea-Lysine (Glu-urea-Lys), Glu-urea-Ornithine (Glu-urea-Orn), Glu-urea-Glutamine (Glu-urea-Gln) and Glu-urea-Asparagine (Glu-urea-Asn) were successfully synthesized, and their structures were confirmed to be as desired using nuclear magnetic resonance spectroscopy and mass spectrometry. An affinity assay was performed to detect the affinity between the various compounds and PSMA expressed from the prostate cancer LNCap cell line. Glu-urea-Gln had the highest affinity to PSMA, followed by Glu-urea-Asn, Glu-urea-Orn and Glu-urea-Lys. In conclusion, the present study demonstrated that Glu-urea-R specifically binds PSMA expressed in the LNCap cell line and inhibits its activity. PMID:27446384

  5. The Monitoring and Affinity Purification of Proteins Using Dual-Tags with Tetracysteine Motifs

    SciTech Connect

    Giannone, Richard J; Liu, Yie; Wang, Yisong

    2009-01-01

    Identification and characterization of protein-protein interaction networks is essential for the elucidation of biochemical mechanisms and cellular function. Affinity purification in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a very powerful tactic for the identification of specific protein-protein interactions. In this chapter we describe a comprehensive methodology that utilizes our recently developed dual-tag affinity purification system for the enrichment and identification of mammalian protein complexes. The protocol covers a series of separate but sequentially related techniques focused on the facile monitoring and purification of a dual-tagged protein of interest and its interacting partners via a system built with tetracysteine motifs and various combinations of affinity tags. Using human telomeric repeat binding factor 2 (TRF2) as an example, we have demonstrated the power of the system in terms of bait protein recovery after dual-tag affinity purification, detection of bait protein subcellular localization and expression, and successful identification of known and potentially novel TRF2 interacting proteins. Although the protocol described here has been optimized for the identification and characterization of TRF2-associated proteins, it is, in principle, applicable to the study of any other mammalian protein complexes that may be of interest to the research community.

  6. Proteomic analysis of Trypanosoma cruzi developmental stages using isotope-coded affinity tag reagents.

    PubMed

    Paba, Jaime; Ricart, Carlos A O; Fontes, Wagner; Santana, Jaime M; Teixeira, Antonio R L; Marchese, Jason; Williamson, Brian; Hunt, Tony; Karger, Barry L; Sousa, Marcelo V

    2004-01-01

    Comparative proteome analysis of developmental stages of the human pathogen Trypanosoma cruzi was carried out by isotope-coded affinity tag technology (ICAT) associated with liquid cromatography-mass spectrometry peptide sequencing (LC-MS/MS). Protein extracts of the protozoan trypomastigote and amastigote stages were labeled with heavy (D8) and light (D0) ICAT reagents and subjected to cation exchange and avidin affinity chromatographies followed by LC-MS/MS analysis. High confidence sequence information and expression levels for 41 T. cruzi polypeptides, including metabolic enzymes, paraflagellar rod components, tubulins, and heat-shock proteins were reported. Twenty-nine proteins displayed similar levels of expression in both forms of the parasite, nine proteins presented higher levels in trypomastigotes, whereas three were more expressed in amastigotes.

  7. X-ray Spectrometry.

    ERIC Educational Resources Information Center

    Markowicz, Andrzej A.; Van Grieken, Rene E.

    1984-01-01

    Provided is a selective literature survey of X-ray spectrometry from late 1981 to late 1983. Literature examined focuses on: excitation (photon and electron excitation and particle-induced X-ray emission; detection (wavelength-dispersive and energy-dispersive spectrometry); instrumentation and techniques; and on such quantitative analytical…

  8. On modality and complexity of affine embeddings

    SciTech Connect

    Arzhantsev, I V

    2001-08-31

    Let G be a reductive algebraic group and let H be a reductive subgroup of G. The modality of a G-variety X is the largest number of the parameters in a continuous family of G-orbits in X. A precise formula for the maximum value of the modality over all affine embeddings of the homogeneous space G/H is obtained.

  9. Modern affinity reagents: Recombinant antibodies and aptamers.

    PubMed

    Groff, Katherine; Brown, Jeffrey; Clippinger, Amy J

    2015-12-01

    Affinity reagents are essential tools in both basic and applied research; however, there is a growing concern about the reproducibility of animal-derived monoclonal antibodies. The need for higher quality affinity reagents has prompted the development of methods that provide scientific, economic, and time-saving advantages and do not require the use of animals. This review describes two types of affinity reagents, recombinant antibodies and aptamers, which are non-animal technologies that can replace the use of animal-derived monoclonal antibodies. Recombinant antibodies are protein-based reagents, while aptamers are nucleic-acid-based. In light of the scientific advantages of these technologies, this review also discusses ways to gain momentum in the use of modern affinity reagents, including an update to the 1999 National Academy of Sciences monoclonal antibody production report and federal incentives for recombinant antibody and aptamer efforts. In the long-term, these efforts have the potential to improve the overall quality and decrease the cost of scientific research.

  10. Validation of affinity reagents using antigen microarrays.

    PubMed

    Sjöberg, Ronald; Sundberg, Mårten; Gundberg, Anna; Sivertsson, Asa; Schwenk, Jochen M; Uhlén, Mathias; Nilsson, Peter

    2012-06-15

    There is a need for standardised validation of affinity reagents to determine their binding selectivity and specificity. This is of particular importance for systematic efforts that aim to cover the human proteome with different types of binding reagents. One such international program is the SH2-consortium, which was formed to generate a complete set of renewable affinity reagents to the SH2-domain containing human proteins. Here, we describe a microarray strategy to validate various affinity reagents, such as recombinant single-chain antibodies, mouse monoclonal antibodies and antigen-purified polyclonal antibodies using a highly multiplexed approach. An SH2-specific antigen microarray was designed and generated, containing more than 6000 spots displayed by 14 identical subarrays each with 406 antigens, where 105 of them represented SH2-domain containing proteins. Approximately 400 different affinity reagents of various types were analysed on these antigen microarrays carrying antigens of different types. The microarrays revealed not only very detailed specificity profiles for all the binders, but also showed that overlapping target sequences of spotted antigens were detected by off-target interactions. The presented study illustrates the feasibility of using antigen microarrays for integrative, high-throughput validation of various types of binders and antigens.

  11. Stabilization of the Motion of Affine Systems

    NASA Astrophysics Data System (ADS)

    Babenko, E. A.; Martynyuk, A. A.

    2016-07-01

    Sufficient conditions for the stability of a nonlinear affine system subject to interval initial conditions are established. These conditions are based on new estimates of the norms of the solutions of the systems of perturbed equations of motion. This stabilization method is used to analyze an electromechanical system with permanent magnet

  12. Fan Affinity Laws from a Collision Model

    ERIC Educational Resources Information Center

    Bhattacharjee, Shayak

    2012-01-01

    The performance of a fan is usually estimated using hydrodynamical considerations. The calculations are long and involved and the results are expressed in terms of three affinity laws. In this paper we use kinetic theory to attack this problem. A hard sphere collision model is used, and subsequently a correction to account for the flow behaviour…

  13. Vygotsky's and Buber's Pedagogical Perspectives: Some Affinities

    ERIC Educational Resources Information Center

    Bartholo, Roberto; Tunes, Elizabeth; Tacca, Maria Carmen Villela Rosa

    2010-01-01

    The purpose of this paper is to examine the dialogical and creative character of pedagogic work by analyzing the affinities between Martin Buber's "I-Thou relation" and Lev Semenovich Vygotsky's "Zone of Proximal Development". Backed up by empirical studies on the teacher-student relation, we understand that education can only result in students'…

  14. Evidence of land plant affinity for the Devonian fossil Protosalvinia (Foerstia)

    USGS Publications Warehouse

    Romankiw, L.A.; Hatcher, P.G.; Roen, J.B.

    1988-01-01

    The Devonian plant fossil Protosalvinia (Foerstia) has been examined by solid-state 13C nuclear magnetic resonance spectroscopy (NMR) and pyrolysis-gas chromatography-mass spectrometry (PY-GC-MS). Results of these studies reveal that the chemical structure of Protosalvinia is remarkably similar to that of coalified wood. A well-defined phenolic carbon peak in the NMR spectra and the appearance of phenol and alkylated phenols in pyrolysis products are clearly indicative of lignin-like compounds. These data represent significant new information on the chemical nature of Protosalvinia and provide the first substantial organic geochemical evidence for land plant affinity. -Authors

  15. Evaluation of SDS depletion using an affinity spin column and IMS-MS detection

    SciTech Connect

    Hengel, Shawna M.; Floyd, Erica A.; Baker, Erin Shammel; Zhao, Rui; Wu, Si; Pasa-Tolic, Ljiljana

    2012-11-01

    While the use of detergents is necessary for a variety of protein isolation preparation protocols, often prior to mass spectral (MS) analysis, they are not compatible with MS analysis due to ion suppression and adduct formation. This manuscript describes optimization of detergent removal, using commercially available SDS depletion spin columns containing an affinity resin, providing for both increased protein recovery and thorough SDS removal. Ion mobility spectrometry coupled with mass spectrometry (IMS-MS) allowed for a concurrent analysis of both analyte and detergent. In the case of both proteins and peptides, higher detergent concentrations than previously reported provided an increase of sample recovery; however there was a limit as SDS was detected by IMS-MS at higher levels of SDS indicating incomplete detergent depletion. The results also suggest optimal conditions for SDS removal are dependent on the sample concentration. Overall, this study provides a useful guide for proteomic studies where SDS is required for efficient sample preparation.

  16. Affinity-based methods in drug-target discovery.

    PubMed

    Rylova, Gabriela; Ozdian, Tomas; Varanasi, Lakshman; Soural, Miroslav; Hlavac, Jan; Holub, Dusan; Dzubak, Petr; Hajduch, Marian

    2015-01-01

    Target discovery using the molecular approach, as opposed to the more traditional systems approach requires the study of the cellular or biological process underlying a condition or disease. The approaches that are employed by the "bench" scientist may be genetic, genomic or proteomic and each has its rightful place in the drug-target discovery process. Affinity-based proteomic techniques currently used in drug-discovery draw upon several disciplines, synthetic chemistry, cell-biology, biochemistry and mass spectrometry. An important component of such techniques is the probe that is specifically designed to pick out a protein or set of proteins from amongst the varied thousands in a cell lysate. A second component, that is just as important, is liquid-chromatography tandem massspectrometry (LC-MS/MS). LC-MS/MS and the supporting theoretical framework has come of age and is the tool of choice for protein identification and quantification. These proteomic tools are critical to maintaining the drug-candidate supply, in the larger context of drug discovery.

  17. Methods for determining the genetic affinity of microorganisms and viruses

    NASA Technical Reports Server (NTRS)

    Fox, George E. (Inventor); Willson, III, Richard C. (Inventor); Zhang, Zhengdong (Inventor)

    2012-01-01

    Selecting which sub-sequences in a database of nucleic acid such as 16S rRNA are highly characteristic of particular groupings of bacteria, microorganisms, fungi, etc. on a substantially phylogenetic tree. Also applicable to viruses comprising viral genomic RNA or DNA. A catalogue of highly characteristic sequences identified by this method is assembled to establish the genetic identity of an unknown organism. The characteristic sequences are used to design nucleic acid hybridization probes that include the characteristic sequence or its complement, or are derived from one or more characteristic sequences. A plurality of these characteristic sequences is used in hybridization to determine the phylogenetic tree position of the organism(s) in a sample. Those target organisms represented in the original sequence database and sufficient characteristic sequences can identify to the species or subspecies level. Oligonucleotide arrays of many probes are especially preferred. A hybridization signal can comprise fluorescence, chemiluminescence, or isotopic labeling, etc.; or sequences in a sample can be detected by direct means, e.g. mass spectrometry. The method's characteristic sequences can also be used to design specific PCR primers. The method uniquely identifies the phylogenetic affinity of an unknown organism without requiring prior knowledge of what is present in the sample. Even if the organism has not been previously encountered, the method still provides useful information about which phylogenetic tree bifurcation nodes encompass the organism.

  18. Mass spectrometry innovations in drug discovery and development.

    PubMed

    Papac, D I; Shahrokh, Z

    2001-02-01

    This review highlights the many roles mass spectrometry plays in the discovery and development of new therapeutics by both the pharmaceutical and the biotechnology industries. Innovations in mass spectrometer source design, improvements to mass accuracy, and implementation of computer-controlled automation have accelerated the purification and characterization of compounds derived from combinatorial libraries, as well as the throughput of pharmacokinetics studies. The use of accelerator mass spectrometry, chemical reaction interface-mass spectrometry and continuous flow-isotope ratio mass spectrometry are promising alternatives for conducting mass balance studies in man. To meet the technical challenges of proteomics, discovery groups in biotechnology companies have led the way to development of instruments with greater sensitivity and mass accuracy (e.g., MALDI-TOF, ESI-Q-TOF, Ion Trap), the miniaturization of separation techniques and ion sources (e.g., capillary HPLC and nanospray), and the utilization of bioinformatics. Affinity-based methods coupled to mass spectrometry are allowing rapid and selective identification of both synthetic and biological molecules. With decreasing instrument cost and size and increasing reliability, mass spectrometers are penetrating both the manufacturing and the quality control arenas. The next generation of technologies to simplify the investigation of the complex fate of novel pharmaceutical entities in vitro and in vivo will be chip-based approaches coupled with mass spectrometry.

  19. Optimal Affine-Invariant Point Matching

    NASA Astrophysics Data System (ADS)

    Costa, Mauro S.; Haralick, Robert M.; Phillips, Tsaiyun I.; Shapiro, Linda G.

    1989-03-01

    The affine-transformation matching scheme proposed by Hummel and Wolfson (1988) is very efficient in a model-based matching system, not only in terms of the computational complexity involved, but also in terms of the simplicity of the method. This paper addresses the implementation of the affine-invariant point matching, applied to the problem of recognizing and determining the pose of sheet metal parts. It points out errors that can occur with this method due to quantization, stability, symmetry, and noise problems. By beginning with an explicit noise model which the Hummel and Wolfson technique lacks, we can derive an optimal approach which overcomes these problems. We show that results obtained with the new algorithm are clearly better than the results from the original method.

  20. Affinity Chromatography in Nonionic Detergent Solutions

    NASA Astrophysics Data System (ADS)

    Robinson, Jack B.; Strottmann, James M.; Wick, Donald G.; Stellwagen, Earle

    1980-10-01

    Anionic dye affinity chromatography is commonly unproductive in the presence of nonionic detergents used to extract particulate proteins. Using lactate dehydrogenase as a model protein, Cibacron blue F3GA as a model dye, and Triton X-100 as a model detergent, we find that the dye is encapsulated in nonionic detergent micelles, rendering the dye incapable of ligation with the enzyme. However, the dye can be liberated from the micelles without altering the nonionic detergent concentration by addition of an anionic detergent, such as deoxycholate or sodium dodecyl sulfate, forming mixed anionic/nonionic micelles that displace the anionic dye. Encapsulation of the anionic detergents prevents their activity as protein denaturants. These observations have been successfully translated to the dye affinity chromatography of a detergent extract of brain particulate cyclic nucleotide phosphodiesterase.

  1. Negative affinity X-ray photocathodes

    NASA Technical Reports Server (NTRS)

    Vanspeybroeck, L.; Kellogg, E.; Murray, S.; Duckett, S.

    1974-01-01

    A new X-ray image intensifier is described. The device should eventually have a quantum efficiency which is an order of magnitude greater than that of presently available high spatial resolution X-ray detectors, such as microchannel plates. The new intesifier is based upon a GaAs crystal photocathode which is activated to achieve negative electron affinity. Details concerning the detector concept are discussed together with the theoretical relations involved, X-ray data, and optical data.

  2. On constructing purely affine theories with matter

    NASA Astrophysics Data System (ADS)

    Cervantes-Cota, Jorge L.; Liebscher, D.-E.

    2016-08-01

    We explore ways to obtain the very existence of a space-time metric from an action principle that does not refer to it a priori. Although there are reasons to believe that only a non-local theory can viably achieve this goal, we investigate here local theories that start with Schrödinger's purely affine theory (Schrödinger in Space-time structure. Cambridge UP, Cambridge, 1950), where he gave reasons to set the metric proportional to the Ricci curvature aposteriori. When we leave the context of unified field theory, and we couple the non-gravitational matter using some weak equivalence principle, we can show that the propagation of shock waves does not define a lightcone when the purely affine theory is local and avoids the explicit use of the Ricci tensor in realizing the weak equivalence principle. When the Ricci tensor is substituted for the metric, the equations seem to have only a very limited set of solutions. This backs the conviction that viable purely affine theories have to be non-local.

  3. Phosphopeptide Enrichment by Immobilized Metal Affinity Chromatography.

    PubMed

    Thingholm, Tine E; Larsen, Martin R

    2016-01-01

    Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively charged metal ions such as Fe(3+), Ga(3+), Al(3+), Zr(4+), and Ti(4+) has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from nonspecific binding of non-phosphorylated peptides. This problem is mainly caused by highly acidic peptides that also share high binding affinity towards these metal ions. By lowering the pH of the loading buffer nonspecific binding can be reduced significantly, however with the risk of reducing specific binding capacity. After binding, the enriched phosphopeptides are released from the metal ions using alkaline buffers of pH 10-11, EDTA, or phosphate-containing buffers. Here we describe a protocol for IMAC using Fe(3+) for phosphopeptide enrichment. The principles are illustrated on a semi-complex peptide mixture.

  4. Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

    PubMed Central

    Nilvebrant, Johan; Åstrand, Mikael; Georgieva-Kotseva, Maria; Björnmalm, Mattias; Löfblom, John; Hober, Sophia

    2014-01-01

    The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein. PMID:25089830

  5. Cysteine-rich secretory proteins in snake venoms form high affinity complexes with human and porcine beta-microseminoproteins.

    PubMed

    Hansson, Karin; Kjellberg, Margareta; Fernlund, Per

    2009-08-01

    BETA-microseminoprotein (MSP), a 10 kDa protein in human seminal plasma, binds human cysteine-rich secretory protein-3 (CRISP-3) with high affinity. CRISP-3 is a member of the family of CRISPs, which are widespread among animals. In this work we show that human as well as porcine MSP binds catrin, latisemin, pseudecin, and triflin, which are CRISPs present in the venoms of the snakes Crotalus atrox, Laticauda semifasciata, Pseudechis porphyriacus, and Trimeresurus flavoviridis, respectively. The CRISPs were purified from the venoms by affinity chromatography on a human MSP column and their identities were settled by gel electrophoresis and mass spectrometry. Their interactions with human and porcine MSPs were studied with size exclusion chromatography and surface plasmon resonance measurements. The binding affinities at 25 degrees C were between 10(-10)M and 10(-7)M for most of the interactions, with higher affinities for the interactions with porcine MSP compared to human MSP and with Elapidae CRISPs compared to Viperidae CRISPs. The high affinities of the bindings in spite of the differences in amino acid sequence between the MSPs as well as between the CRISPs indicate that the binding is tolerant to amino acid sequence variation and raise the question how universal this cross-species reaction between MSPs and CRISPs is.

  6. Early Detection of Cancer by Affinity Mass Spectrometry-Set Aside funds — EDRN Public Portal

    Cancer.gov

    A.   RATIONALE The recent introduction of multiple reaction monitoring capabilities offers unprecedented capability to the research arsenal available to protein based biomarker discovery. Specific to the discovery process this technology offers an ability to monitor specific protein changes in concentration and/or post-translational modification. The ability to accurately confirm specific biomarkers in a sensitive and reproducible manner is critical to the confirmation and pre-validation process. We are proposing two collaborative studies that promise to develop Multiple Reaction Monitoring (MRM) work flows for the biomarker scientific community and specifically for EDRN. B.   GOALS The overall goal for this proposal is the identification of protein biomarkers that can be associated with prostate cancer detection. The underlying goal is the application of a novel technological approach aided by MRM toward biomarker discovery. An additional goal will be the dissemination of knowledge gained from these studies EDRN wide.

  7. Integrin avidity regulation: are changes in affinity and conformation underemphasized?

    PubMed

    Carman, Christopher V; Springer, Timothy A

    2003-10-01

    Integrins play critical roles in development, wound healing, immunity and cancer. Central to their function is their unique ability to modulate dynamically their adhesiveness through both affinity- and valency-based mechanisms. Recent advances have shed light on the structural basis for affinity regulation and on the signaling mechanisms responsible for both affinity and valency modes of regulation.

  8. Functional phosphoproteomic mass spectrometry-based approaches

    PubMed Central

    2012-01-01

    Mass Spectrometry (MS)-based phosphoproteomics tools are crucial for understanding the structure and dynamics of signaling networks. Approaches such as affinity purification followed by MS have also been used to elucidate relevant biological questions in health and disease. The study of proteomes and phosphoproteomes as linked systems, rather than research studies of individual proteins, are necessary to understand the functions of phosphorylated and un-phosphorylated proteins under spatial and temporal conditions. Phosphoproteome studies also facilitate drug target protein identification which may be clinically useful in the near future. Here, we provide an overview of general principles of signaling pathways versus phosphorylation. Likewise, we detail chemical phosphoproteomic tools, including pros and cons with examples where these methods have been applied. In addition, basic clues of electrospray ionization and collision induced dissociation fragmentation are detailed in a simple manner for successful phosphoproteomic clinical studies. PMID:23369623

  9. Immobilized Metal Affinity Chromatography Coupled to Multiple Reaction Monitoring Enables Reproducible Quantification of Phospho-signaling*

    PubMed Central

    Kennedy, Jacob J.; Yan, Ping; Zhao, Lei; Ivey, Richard G.; Voytovich, Uliana J.; Moore, Heather D.; Lin, Chenwei; Pogosova-Agadjanyan, Era L.; Stirewalt, Derek L.; Reding, Kerryn W.; Whiteaker, Jeffrey R.; Paulovich, Amanda G.

    2016-01-01

    A major goal in cell signaling research is the quantification of phosphorylation pharmacodynamics following perturbations. Traditional methods of studying cellular phospho-signaling measure one analyte at a time with poor standardization, rendering them inadequate for interrogating network biology and contributing to the irreproducibility of preclinical research. In this study, we test the feasibility of circumventing these issues by coupling immobilized metal affinity chromatography (IMAC)-based enrichment of phosphopeptides with targeted, multiple reaction monitoring (MRM) mass spectrometry to achieve precise, specific, standardized, multiplex quantification of phospho-signaling responses. A multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay targeting phospho-analytes responsive to DNA damage was configured, analytically characterized, and deployed to generate phospho-pharmacodynamic curves from primary and immortalized human cells experiencing genotoxic stress. The multiplexed assays demonstrated linear ranges of ≥3 orders of magnitude, median lower limit of quantification of 0.64 fmol on column, median intra-assay variability of 9.3%, median inter-assay variability of 12.7%, and median total CV of 16.0%. The multiplex immobilized metal affinity chromatography- multiple reaction monitoring assay enabled robust quantification of 107 DNA damage-responsive phosphosites from human cells following DNA damage. The assays have been made publicly available as a resource to the community. The approach is generally applicable, enabling wide interrogation of signaling networks. PMID:26621847

  10. Theoretical and Experimental Determination of the Proton Affinity of (CF3CH2)2O

    NASA Technical Reports Server (NTRS)

    Zehe, Michael J.; Ball, David W.

    1998-01-01

    We report the experimental determination of the proton affinity of the molecule (CF3CH2)2O using chemical ionization mass spectrometry, and we compare it to the theoretical value obtained for protonation at the oxygen atom using the calculational methodology (MP2/6-31G**//MP2/3-21G). The proton affinity for this molecule as measured by bracketing experiments was between 724 kJ/mole and 741 kJ/mole. Ab initio (MP2/6-31G**//MP2/3-21G) calculations yield a value of about 729 kJ/mole, in agreement with the chemical ionization experiments. The results of these and related calculations suggest that the (MP2/6-31G**//MP2/3-21G) methodology is acceptable for estimating the proton affinities of partially-and fully-fluorinated methyl and ethyl ethers. We submit that any conclusions about the chemistry of fluoroether polymer lubricants based on their basicity can also be predicted reliably with such calculations.

  11. Affinity Purification and Characterization of a G-Protein Coupled Receptor, Saccharomyces cerevisiae Ste2p

    SciTech Connect

    Lee, Byung-Kwon; Jung, Kyung-Sik; Son, Cagdas D; Kim, Heejung; Verberkmoes, Nathan C; Arshava, Boris; Naider, Fred; Becker, Jeffrey Marvin

    2007-01-01

    We present a rare example of a biologically active G protein coupled receptor (GPCR) whose purity and identity were verified by mass spectrometry after being purified to near homogeneity from its native system. An overexpression vector was constructed to encode the Saccharomyces cerevisiae GPCR -factor receptor (Ste2p, the STE2 gene product) containing a 9-amino acid sequence of rhodopsin that served as an epitope/affinity tag. In the construct, two glycosylation sites and two cysteine residues were removed to aid future structural and functional studies. The receptor was expressed in yeast cells and was detected as a single band in a western blot indicating the absence of glycosylation. Tests of the epitope-tagged, mutated receptor showed it maintained its full biological activity. For extraction of Ste2p, yeast membranes were solubilized with 0.5 % n-dodecyl maltoside (DM). Approximately 120 g of purified -factor receptor was obtained per liter of culture by single-step affinity chromatography using a monoclonal antibody to the rhodopsin epitope. The binding affinity (Kd) of the purified -factor receptor in DM micelles was 28 nM as compared to Kd = 12.7 nM for Ste2p in cell membranes, and approximately 40 % of the purified receptor was correctly folded as judged by ligand saturation binding. About 50 % of the receptor sequence was retrieved from MALDITOF and nanospray mass spectrometry after CNBr digestion of the purified receptor. The methods described will enable structural studies of the -factor receptor and may provide an efficient technique to purify other GPCRs that have been functionally expressed in yeast.

  12. Latest European coelacanth shows Gondwanan affinities.

    PubMed

    Cavin, Lionel; Forey, Peter L; Buffetaut, Eric; Tong, Haiyan

    2005-06-22

    The last European fossil occurrence of a coelacanth is from the Mid-Cretaceous of the English Chalk (Turonian, 90 million years ago). Here, we report the discovery of a coelacanth from Late Cretaceous non-marine rocks in southern France. It consists of a left angular bone showing structures that imply close phylogenetic affinities with some extinct Mawsoniidae. The closest relatives are otherwise known from Cretaceous continental deposits of southern continents and suggest that the dispersal of freshwater organisms from Africa to Europe occurred in the Late Cretaceous.

  13. On the electron affinity of Be2

    NASA Technical Reports Server (NTRS)

    Bauschlicher, C. W., Jr.; Partridge, H.

    1984-01-01

    Calculations of the electron affinity (EA) of Be2 using a large Slater-type orbital basis set and extensive correlation based upon a CASSCF reference are reported. The adiabatic EAs are estimated to be 0.44 eV for the 2Sigma sub g(+) state and 0.56 eV for the 2Pi sub u state. The extra electron attaches into an empty bonding orbital, causing a shortening of the bond length and an increase in omega(e). The D(e) of the 2Pi sub u state of Be2 is six times as large as the D(e) of Be2.

  14. On the structure of self-affine convex bodies

    SciTech Connect

    Voynov, A S

    2013-08-31

    We study the structure of convex bodies in R{sup d} that can be represented as a union of their affine images with no common interior points. Such bodies are called self-affine. Vallet's conjecture on the structure of self-affine bodies was proved for d = 2 by Richter in 2011. In the present paper we disprove the conjecture for all d≥3 and derive a detailed description of self-affine bodies in R{sup 3}. Also we consider the relation between properties of self-affine bodies and functional equations with a contraction of an argument. Bibliography: 10 titles.

  15. Forensic Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Hoffmann, William D.; Jackson, Glen P.

    2015-07-01

    Developments in forensic mass spectrometry tend to follow, rather than lead, the developments in other disciplines. Examples of techniques having forensic potential born independently of forensic applications include ambient ionization, imaging mass spectrometry, isotope ratio mass spectrometry, portable mass spectrometers, and hyphenated chromatography-mass spectrometry instruments, to name a few. Forensic science has the potential to benefit enormously from developments that are funded by other means, if only the infrastructure and personnel existed to adopt, validate, and implement the new technologies into casework. Perhaps one unique area in which forensic science is at the cutting edge is in the area of chemometrics and the determination of likelihood ratios for the evaluation of the weight of evidence. Such statistical techniques have been developed most extensively for ignitable-liquid residue analyses and isotope ratio analysis. This review attempts to capture the trends, motivating forces, and likely impact of developing areas of forensic mass spectrometry, with the caveat that none of this research is likely to have any real impact in the forensic community unless: (a) The instruments developed are turned into robust black boxes with red and green lights for positives and negatives, respectively, or (b) there are PhD graduates in the workforce who can help adopt these sophisticated techniques.

  16. Forensic Mass Spectrometry.

    PubMed

    Hoffmann, William D; Jackson, Glen P

    2015-01-01

    Developments in forensic mass spectrometry tend to follow, rather than lead, the developments in other disciplines. Examples of techniques having forensic potential born independently of forensic applications include ambient ionization, imaging mass spectrometry, isotope ratio mass spectrometry, portable mass spectrometers, and hyphenated chromatography-mass spectrometry instruments, to name a few. Forensic science has the potential to benefit enormously from developments that are funded by other means, if only the infrastructure and personnel existed to adopt, validate, and implement the new technologies into casework. Perhaps one unique area in which forensic science is at the cutting edge is in the area of chemometrics and the determination of likelihood ratios for the evaluation of the weight of evidence. Such statistical techniques have been developed most extensively for ignitable-liquid residue analyses and isotope ratio analysis. This review attempts to capture the trends, motivating forces, and likely impact of developing areas of forensic mass spectrometry, with the caveat that none of this research is likely to have any real impact in the forensic community unless: (a) The instruments developed are turned into robust black boxes with red and green lights for positives and negatives, respectively, or (b) there are PhD graduates in the workforce who can help adopt these sophisticated techniques.

  17. Affinity filtration coupled with capillary-based affinity purification for the isolation of protein complexes.

    PubMed

    Qureshi, M S; Sheikh, Q I; Hill, R; Brown, P E; Dickman, M J; Tzokov, S B; Rice, D W; Gjerde, D T; Hornby, D P

    2013-08-01

    The isolation of complex macromolecular assemblies at the concentrations required for structural analysis represents a major experimental challenge. Here we present a method that combines the genetic power of site-specific recombination in order to selectively "tag" one or more components of a protein complex with affinity-based rapid filtration and a final step of capillary-based enrichment. This modified form of tandem affinity purification produces highly purified protein complexes at high concentrations in a highly efficient manner. The application of the method is demonstrated for the yeast Arp2/3 heptameric protein complex involved in mediating reorganization of the actin cytoskeleton.

  18. Extraction of haemoglobin from human blood by affinity precipitation using a haptoglobin-based stimuli-responsive affinity macroligand.

    PubMed

    Stocker-Majd, Gisela; Hilbrig, Frank; Freitag, Ruth

    2008-06-13

    Affinity precipitation was compared to affinity chromatography and batch adsorption as the final purification step in a protocol for the isolation of haemoglobin from human blood. Haptoglobin was the affinity ligand. The first steps on the process were realized by traditional methods (lyses of red blood cells followed by ammonium sulphate precipitation). For affinity chromatography (and batch adsorption) the ligand was linked to Sepharose, for affinity precipitation to a thermoresponsive polymer, namely poly(N-isopropylacrylamide). Five haptoglobin-poly(N-isopropylacrylamide) bioconjugates (affinity macroligands) were constructed with different polymer: haptoglobin-coupling ratios. Conjugation of haptoglobin to the soluble poly(N-isopropylacrylamide) apparently does not change the interaction thermodynamics with haemoglobin, as the haemoglobin binding constants calculated by a Scatchard analysis for the affinity macroligand were of the same order of magnitude as those described in the literature for the haemoglobin-haptoglobin complex in solution. Two elution protocols were used for haemoglobin release from the various affinity materials, one at pH 2, the other with 5 M urea at pH 11. Both affinity chromatography and affinity precipitation yielded a pure haemoglobin of high quality. Compared to the affinity chromatography, affinity precipitation showed a significantly higher ligand efficiency (ratio of the experimental capacity to the theoretical one). The method thus makes better use of the expensive affinity ligands. As affinity precipitation only requires small temperature changes to bring about precipitation/redissolution of the affinity complexes and a centrifugation step for recovery of the precipitate, the method in addition has advantages in term of scalability and simplicity.

  19. Etalon Array Reconstructive Spectrometry

    NASA Astrophysics Data System (ADS)

    Huang, Eric; Ma, Qian; Liu, Zhaowei

    2017-01-01

    Compact spectrometers are crucial in areas where size and weight may need to be minimized. These types of spectrometers often contain no moving parts, which makes for an instrument that can be highly durable. With the recent proliferation in low-cost and high-resolution cameras, camera-based spectrometry methods have the potential to make portable spectrometers small, ubiquitous, and cheap. Here, we demonstrate a novel method for compact spectrometry that uses an array of etalons to perform spectral encoding, and uses a reconstruction algorithm to recover the incident spectrum. This spectrometer has the unique capability for both high resolution and a large working bandwidth without sacrificing sensitivity, and we anticipate that its simplicity makes it an excellent candidate whenever a compact, robust, and flexible spectrometry solution is needed.

  20. Etalon Array Reconstructive Spectrometry

    PubMed Central

    Huang, Eric; Ma, Qian; Liu, Zhaowei

    2017-01-01

    Compact spectrometers are crucial in areas where size and weight may need to be minimized. These types of spectrometers often contain no moving parts, which makes for an instrument that can be highly durable. With the recent proliferation in low-cost and high-resolution cameras, camera-based spectrometry methods have the potential to make portable spectrometers small, ubiquitous, and cheap. Here, we demonstrate a novel method for compact spectrometry that uses an array of etalons to perform spectral encoding, and uses a reconstruction algorithm to recover the incident spectrum. This spectrometer has the unique capability for both high resolution and a large working bandwidth without sacrificing sensitivity, and we anticipate that its simplicity makes it an excellent candidate whenever a compact, robust, and flexible spectrometry solution is needed. PMID:28074883

  1. Quantification of hydrophobic interaction affinity of colloids

    NASA Astrophysics Data System (ADS)

    Saini, G.; Nasholm, N.; Wood, B. D.

    2009-12-01

    Colloids play an important role in a wide variety of disciplines, including water and wastewater treatment, subsurface transport of metals and organic contaminants, migration of fines in oil reservoirs, biocolloid (virus and bacteria) transport in subsurface, and are integral to laboratory transport studies. Although the role of hydrophobicity in adhesion and transport of colloids, particularly bacteria, is well known; there is scarcity of literature regarding hydrophobicity measurement of non-bacterial colloids and other micron-sized particles. Here we detail an experimental approach based on differential partitioning of colloids between two liquid phases (hydrocarbon and buffer) as a measure of the hydrophobic interaction affinity of colloids. This assay, known as Microbial adhesion to hydrocarbons or MATH, is frequently used in microbiology and bacteriology for quantifying the hydrophobicity of microbes. Monodispersed colloids and particles, with sizes ranging from 1 micron to 33 micron, were used for the experiments. A range of hydrophobicity values were observed for different particles. The hydrophobicity results are also verified against water contact angle measurements of these particles. This liquid-liquid partitioning assay is quick, easy-to-perform and requires minimal instrumentation. Estimation of the hydrophobic interaction affinity of colloids would lead to a better understanding of their adhesion to different surfaces and subsequent transport in porous media.

  2. Fatigue damage prognosis using affine arithmetic

    NASA Astrophysics Data System (ADS)

    Gbaguidi, Audrey; Kim, Daewon

    2014-02-01

    Among the essential steps to be taken in structural health monitoring systems, damage prognosis would be the field that is least investigated due to the complexity of the uncertainties. This paper presents the possibility of using Affine Arithmetic for uncertainty propagation of crack damage in damage prognosis. The structures examined are thin rectangular plates made of titanium alloys with central mode I cracks and a composite plate with an internal delamination caused by mixed mode I and II fracture modes, under a harmonic uniaxial loading condition. The model-based method for crack growth rates are considered using the Paris Erdogan law model for the isotropic plates and the delamination growth law model proposed by Kardomateas for the composite plate. The parameters for both models are randomly taken and their uncertainties are considered as defined by an interval instead of a probability distribution. A Monte Carlo method is also applied to check whether Affine Arithmetic (AA) leads to tight bounds on the lifetime of the structure.

  3. Affine conformal vectors in space-time

    NASA Astrophysics Data System (ADS)

    Coley, A. A.; Tupper, B. O. J.

    1992-05-01

    All space-times admitting a proper affine conformal vector (ACV) are found. By using a theorem of Hall and da Costa, it is shown that such space-times either (i) admit a covariantly constant vector (timelike, spacelike, or null) and the ACV is the sum of a proper affine vector and a conformal Killing vector or (ii) the space-time is 2+2 decomposable, in which case it is shown that no ACV can exist (unless the space-time decomposes further). Furthermore, it is proved that all space-times admitting an ACV and a null covariantly constant vector (which are necessarily generalized pp-wave space-times) must have Ricci tensor of Segré type {2,(1,1)}. It follows that, among space-times admitting proper ACV, the Einstein static universe is the only perfect fluid space-time, there are no non-null Einstein-Maxwell space-times, and only the pp-wave space-times are representative of null Einstein-Maxwell solutions. Otherwise, the space-times can represent anisotropic fluids and viscous heat-conducting fluids, but only with restricted equations of state in each case.

  4. Ion Mobility Spectrometry (IMS) and Mass Spectrometry

    SciTech Connect

    Shvartsburg, Alexandre A.

    2010-04-20

    In a media of finite viscosity, the Coulomb force of external electric field moves ions with some terminal speed. This dynamics is controlled by “mobility” - a property of the interaction potential between ions and media molecules. This fact has been used to separate and characterize gas-phase ions in various modes of ion mobility spectrometry (IMS) developed since 1970. Commercial IMS devices were introduced in 1980-s for field detection of volatile traces such as explosives and chemical warfare agents. Coupling to soft-ionization sources, mass spectrometry (MS), and chromatographic methods in 1990-s had allowed IMS to handle complex samples, enabling new applications in biological and environmental analyses, nanoscience, and other areas. Since 2003, the introduction of commercial systems by major instrument vendors started bringing the IMS/MS capability to broad user community. The other major development of last decade has been the differential IMS or “field asymmetric waveform IMS” (FAIMS) that employs asymmetric time-dependent electric field to sort ions not by mobility itself, but by the difference between its values in strong and weak electric fields. Coupling of FAIMS to conventional IMS and stacking of conventional IMS stages have enabled two-dimensional separations that dramatically expand the power of ion mobility methods.

  5. Ambient ionization mass spectrometry

    NASA Astrophysics Data System (ADS)

    Lebedev, A. T.

    2015-07-01

    Ambient ionization mass spectrometry emerged as a new scientific discipline only about ten years ago. A considerable body of information has been reported since that time. Keeping the sensitivity, performance and informativity of classical mass spectrometry methods, the new approach made it possible to eliminate laborious sample preparation procedures and triggered the development of miniaturized instruments to work directly in the field. The review concerns the theoretical foundations and design of ambient ionization methods. Their advantages and drawbacks, as well as prospects for application in chemistry, biology, medicine, environmetal analysis, etc., are discussed. The bibliography includes 194 references.

  6. Affinity Crystallography: A New Approach to Extracting High-Affinity Enzyme Inhibitors from Natural Extracts.

    PubMed

    Aguda, Adeleke H; Lavallee, Vincent; Cheng, Ping; Bott, Tina M; Meimetis, Labros G; Law, Simon; Nguyen, Nham T; Williams, David E; Kaleta, Jadwiga; Villanueva, Ivan; Davies, Julian; Andersen, Raymond J; Brayer, Gary D; Brömme, Dieter

    2016-08-26

    Natural products are an important source of novel drug scaffolds. The highly variable and unpredictable timelines associated with isolating novel compounds and elucidating their structures have led to the demise of exploring natural product extract libraries in drug discovery programs. Here we introduce affinity crystallography as a new methodology that significantly shortens the time of the hit to active structure cycle in bioactive natural product discovery research. This affinity crystallography approach is illustrated by using semipure fractions of an actinomycetes culture extract to isolate and identify a cathepsin K inhibitor and to compare the outcome with the traditional assay-guided purification/structural analysis approach. The traditional approach resulted in the identification of the known inhibitor antipain (1) and its new but lower potency dehydration product 2, while the affinity crystallography approach led to the identification of a new high-affinity inhibitor named lichostatinal (3). The structure and potency of lichostatinal (3) was verified by total synthesis and kinetic characterization. To the best of our knowledge, this is the first example of isolating and characterizing a potent enzyme inhibitor from a partially purified crude natural product extract using a protein crystallographic approach.

  7. Structure of a High-Affinity

    SciTech Connect

    Saphire, E.O.; Montero, M.; Menendez, A.; Houten, N.E.van; Irving, M.B.; Pantophlet, R.; Swick, M.B.; Parren, P.W.H.I.; Burton, D.R.; Scott, J.K.; Wilson, I.A.; /Scripps Res. Inst. /Simon Fraser U. /British Columbia U.

    2007-07-13

    The human antibody b12 recognizes a discontinuous epitope on gp120 and is one of the rare monoclonal antibodies that neutralize a broad range of primary human immunodeficiency virus type 1 (HIV-1) isolates. We previously reported the isolation of B2.1, a dimeric peptide that binds with high specificity to b12 and competes with gp120 for b12 antibody binding. Here, we show that the affinity of B2.1 was improved 60-fold over its synthetic-peptide counterpart by fusing it to the N terminus of a soluble protein. This affinity, which is within an order of magnitude of that of gp120, probably more closely reflects the affinity of the phage-borne peptide. The crystal structure of a complex between Fab of b12 and B2.1 was determined at 1.8 Angstrom resolution. The structural data allowed the differentiation of residues that form critical contacts with b12 from those required for maintenance of the antigenic structure of the peptide, and revealed that three contiguous residues mediate B2.1's critical contacts with b12. This single region of critical contact between the B2.1 peptide and the b12 paratope is unlikely to mimic the discontinuous key binding residues involved in the full b12 epitope for gp120, as previously identified by alanine scanning substitutions on the gp120 surface. These structural observations are supported by experiments that demonstrate that B2.1 is an ineffective immunogenic mimic of the b12 epitope on gp120. Indeed, an extensive series of immunizations with B2.1 in various forms failed to produce gp120 cross-reactive sera. The functional and structural data presented here, however, suggest that the mechanism by which b12 recognizes the two antigens is very different. Here, we present the first crystal structure of peptide bound to an antibody that was originally raised against a discontinuous protein epitope. Our results highlight the challenge of producing immunogens that mimic discontinuous protein epitopes, and the necessity of combining

  8. Modulating Uranium Binding Affinity in Engineered Calmodulin EF-Hand Peptides: Effect of Phosphorylation

    PubMed Central

    Pardoux, Romain; Sauge-Merle, Sandrine; Lemaire, David; Delangle, Pascale; Guilloreau, Luc; Adriano, Jean-Marc; Berthomieu, Catherine

    2012-01-01

    To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T9TKE12 sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from Kd = 25±6 nM to Kd = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (Kd = 0.25±0.06 nM). FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the νas(P-O) and νs(P-O) IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in νas(UO2)2+ vibration (from 923 cm−1 to 908 cm−1) was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH. PMID:22870263

  9. Fourier Transform Mass Spectrometry.

    ERIC Educational Resources Information Center

    Gross, Michael L.; Rempel, Don L.

    1984-01-01

    Discusses the nature of Fourier transform mass spectrometry and its unique combination of high mass resolution, high upper mass limit, and multichannel advantage. Examines its operation, capabilities and limitations, applications (ion storage, ion manipulation, ion chemistry), and future applications and developments. (JN)

  10. Analytical mass spectrometry

    SciTech Connect

    Not Available

    1990-01-01

    This 43rd Annual Summer Symposium on Analytical Chemistry was held July 24--27, 1990 at Oak Ridge, TN and contained sessions on the following topics: Fundamentals of Analytical Mass Spectrometry (MS), MS in the National Laboratories, Lasers and Fourier Transform Methods, Future of MS, New Ionization and LC/MS Methods, and an extra session. (WET)

  11. Analytical mass spectrometry. Abstracts

    SciTech Connect

    Not Available

    1990-12-31

    This 43rd Annual Summer Symposium on Analytical Chemistry was held July 24--27, 1990 at Oak Ridge, TN and contained sessions on the following topics: Fundamentals of Analytical Mass Spectrometry (MS), MS in the National Laboratories, Lasers and Fourier Transform Methods, Future of MS, New Ionization and LC/MS Methods, and an extra session. (WET)

  12. MASS SPECTROMETRY IN ENVIRONMENTAL SCIENCES

    EPA Science Inventory

    This review covers applications of mass spectrometry to the environmental sciences. From the early applications of mass spectrometry to environmental research in the 1960s and 1970s, mass spectrometry has played an important role in aiding our understanding of environmental poll...

  13. The affine cohomology spaces and its applications

    NASA Astrophysics Data System (ADS)

    Fraj, Nizar Ben; Laraiedh, Ismail

    2016-12-01

    We compute the nth cohomology space of the affine Lie superalgebra 𝔞𝔣𝔣(1) on the (1,1)-dimensional real superspace with coefficient in a large class of 𝔞𝔣𝔣(1)-modules M. We apply our results to the module of weight densities and the module of linear differential operators acting on a superspace of weighted densities. This work is the generalization of a result by Basdouri et al. [The linear 𝔞𝔣𝔣(n|1)-invariant differential operators on weighted densities on the superspace ℝ1|n and 𝔞𝔣𝔣(n|1)-relative cohomology, Int. J. Geom. Meth. Mod. Phys. 10 (2013), Article ID: 1320004, 9 pp.

  14. Automatic gesture analysis using constant affine velocity.

    PubMed

    Cifuentes, Jenny; Boulanger, Pierre; Pham, Minh Tu; Moreau, Richard; Prieto, Flavio

    2014-01-01

    Hand human gesture recognition has been an important research topic widely studied around the world, as this field offers the ability to identify, recognize, and analyze human gestures in order to control devices or to interact with computer interfaces. In particular, in medical training, this approach is an important tool that can be used to obtain an objective evaluation of a procedure performance. In this paper, some obstetrical gestures, acquired by a forceps, were studied with the hypothesis that, as the scribbling and drawing movements, they obey the one-sixth power law, an empirical relationship which connects path curvature, torsion, and euclidean velocity. Our results show that obstetrical gestures have a constant affine velocity, which is different for each type of gesture and based on this idea this quantity is proposed as an appropriate classification feature in the hand human gesture recognition field.

  15. Dynamic friction of self-affine surfaces

    NASA Astrophysics Data System (ADS)

    Schmittbuhl, Jean; Vilotte, Jean-Pierre; Roux, Stéphane

    1994-02-01

    We investigate the velocity dependence of the friction between two rigid blocks limited by a self-affine surface such as the one generated by a crack. The upper solid is subjected either to gravity or to an external elastic stiffness, and is driven horizontally at constant velocity, V, while the lower solid is fixed. For low velocities, the apparent friction coefficient is constant. For high velocities, the apparent friction is shown to display a velocity weakening. The weakening can be related to the variation of the mean contact time due to the occurrence of jumps during the motions. The cross-over between these two regimes corresponds to a characteristic velocity which depends on the geometry of the surfaces and on the mean normal force. In the case of simple gravity loading, the velocity dependence of the apparent friction at high velocities is proportional to 1/V^2 where V is the imposed tangential velocity. In the case of external elastic stiffness, two velocity weakening regimes can be identified, the first is identical to the gravity case with a 1/V^2 dependence, the second appears at higher velocities and is characterized by a 1/V variation. The characteristic velocity of this second cross-over depends on the roughness and the elastic stiffness. The statistical distribution of ballistic flight distances is analysed, and is shown to reveal in all cases the self-affinity of the contacting surfaces. Nous analysons la dépendence en vitesse du frottement entre deux solides limités par une surface rugueuse auto-affine comme celle d'une surface de fracture. Le solide supérieur est soumis soit à la gravité, soit à une raideur élastique externe, et est entraîné à vitesse horizontale constante V sur le solide inférieur fixe. A faible vitesse, le coefficient de friction apparent, est constant. A forte vitesse, le coefficient de friction apparent devient inversement proportionnel à la vitesse. Cette dépendance peut être reliée à la variation du temps

  16. Differential Mobility Spectrometry: Preliminary Findings on Determination of Fundamental Constants

    NASA Technical Reports Server (NTRS)

    Limero, Thomas; Cheng, Patti; Boyd, John

    2007-01-01

    The electron capture detector (ECD) has been used for 40+ years (1) to derive fundamental constants such as a compound's electron affinity. Given this historical perspective, it is not surprising that differential mobility spectrometry (DMS) might be used in a like manner. This paper will present data from a gas chromatography (GC)-DMS instrument that illustrates the potential capability of this device to derive fundamental constants for electron-capturing compounds. Potential energy curves will be used to provide possible explanation of the data.

  17. Immunoproteomics using polyclonal antibodies and stable isotope-labeled affinity-purified recombinant proteins.

    PubMed

    Edfors, Fredrik; Boström, Tove; Forsström, Björn; Zeiler, Marlis; Johansson, Henrik; Lundberg, Emma; Hober, Sophia; Lehtiö, Janne; Mann, Matthias; Uhlen, Mathias

    2014-06-01

    The combination of immuno-based methods and mass spectrometry detection has great potential in the field of quantitative proteomics. Here, we describe a new method (immuno-SILAC) for the absolute quantification of proteins in complex samples based on polyclonal antibodies and stable isotope-labeled recombinant protein fragments to allow affinity enrichment prior to mass spectrometry analysis and accurate quantification. We took advantage of the antibody resources publicly available from the Human Protein Atlas project covering more than 80% of all human protein-coding genes. Epitope mapping revealed that a majority of the polyclonal antibodies recognized multiple linear epitopes, and based on these results, a semi-automated method was developed for peptide enrichment using polyclonal antibodies immobilized on protein A-coated magnetic beads. A protocol based on the simultaneous multiplex capture of more than 40 protein targets showed that approximately half of the antibodies enriched at least one functional peptide detected in the subsequent mass spectrometry analysis. The approach was further developed to also generate quantitative data via the addition of heavy isotope-labeled recombinant protein fragment standards prior to trypsin digestion. Here, we show that we were able to use small amounts of antibodies (50 ng per target) in this manner for efficient multiplex analysis of quantitative levels of proteins in a human HeLa cell lysate. The results suggest that polyclonal antibodies generated via immunization of recombinant protein fragments could be used for the enrichment of target peptides to allow for rapid mass spectrometry analysis taking advantage of a substantial reduction in sample complexity. The possibility of building up a proteome-wide resource for immuno-SILAC assays based on publicly available antibody resources is discussed.

  18. Smooth affine shear tight frames: digitization and applications

    NASA Astrophysics Data System (ADS)

    Zhuang, Xiaosheng

    2015-08-01

    In this paper, we mainly discuss one of the recent developed directional multiscale representation systems: smooth affine shear tight frames. A directional wavelet tight frame is generated by isotropic dilations and translations of directional wavelet generators, while an affine shear tight frame is generated by anisotropic dilations, shears, and translations of shearlet generators. These two tight frames are actually connected in the sense that the affine shear tight frame can be obtained from a directional wavelet tight frame through subsampling. Consequently, an affine shear tight frame indeed has an underlying filter bank from the MRA structure of its associated directional wavelet tight frame. We call such filter banks affine shear filter banks, which can be designed completely in the frequency domain. We discuss the digitization of affine shear filter banks and their implementations: the forward and backward digital affine shear transforms. Redundancy rate and computational complexity of digital affine shear transforms are also investigated in this paper. Numerical experiments and comparisons in image/video processing show the advantages of digital affine shear transforms over many other state-of-art directional multiscale representation systems.

  19. A Genome-Inspired DNA Ligand for Affinity Capture of Insulin and Insulin-like Growth Factor-2

    PubMed Central

    Xiao, Junfeng; Carter, Jennifer A.; Frederick, Kimberley A.; McGown, Linda B.

    2009-01-01

    The insulin-linked polymorphic region (ILPR) of the human insulin gene contains tandem repeats of similar G-rich sequences, some of which form intramolecular G-quadruplex structures in vitro. Previous work showed affinity binding of insulin to an intramolecular G-quadruplex formed by ILPR variant a. Here we report on interactions of insulin and the highly homologous insulin-like growth factor 2 (IGF-2) with ILPR variants a, h and i. Circular dichroism indicated intramolecular G-quadruplex formation for variants a and h. Affinity MALDI mass spectrometry and surface plasmon resonance were used to compare protein capture and binding strengths. Insulin and IGF-2 exhibited high binding affinity for variants a and h but not i, indicating the involvement of intramolecular G-quadruplexes. Interaction between insulin and variant a was unique in the appearance of two binding interactions with KD~10−13 M and KD~10−7 M, which was not observed for insulin with variant h (KD~10−8 M) or IGF-2 with either variant (KD’s~10−9 D M). The results provide a basis for design of DNA binding ligands for insulin and IGF-2 and support a new approach to discovery of DNA affinity binding ligands based on genome-inspired sequences rather than the traditional combinatorial selection route to aptamer discovery. PMID:19391177

  20. Noncompetitive affinity assays of glucagon and amylin using mirror-image aptamers as affinity probes.

    PubMed

    Yi, Lian; Wang, Xue; Bethge, Lucas; Klussmann, Sven; Roper, Michael G

    2016-03-21

    The ability to detect picomolar concentrations of glucagon and amylin using fluorescently labeled mirror-image aptamers, so-called Spiegelmers, is demonstrated. Spiegelmers rival the specificity of antibodies and overcome the problem of biostability of natural aptamers in a biological matrix. Using Spiegelmers as affinity probes, noncompetitive capillary electrophoresis affinity assays of glucagon and murine amylin were developed and optimized. The detection limit for glucagon was 6 pM and for amylin was 40 pM. Glucagon-like peptide-1 and -2 did not interfere with the glucagon assay, while the amylin assay showed cross-reactivity to calcitonin gene related peptide. The developed assays were combined with a competitive immunoassay for insulin to measure glucagon, amylin, and insulin secretion from batches of islets after incubation with different glucose concentrations. The development of these assays is an important step towards incorporation into an online measurement system for monitoring dynamic secretion from single islets.

  1. Characterization of naturally-occurring and modified fullerenes by Fourier transform mass spectrometry

    NASA Astrophysics Data System (ADS)

    Hettich, Robert L.; Jin, Changming; Compton, Robert N.; Buseck, Peter R.; Tsipursky, Semeon J.

    1993-10-01

    Fourier transform mass spectrometry (FTMS) employing both laser desorption/ionization and thermal desorption/electron ionization is useful for the detection and structural characterization of fullerenes and chemically-modified fullerenes. Examination of a carbon-rich shungite rock sample from Russia by transmission electron microscopy and FTMS provided evidence of naturally-occurring fullerenes. Ion-molecule reactions can be studied with FTMS to investigate the electron affinities of modified fullerenes. By monitoring charge exchange reactions, the electron affinities of C60Fx (x=44,46) and C70Fy (y=52,54) were found to be substantially higher than the values for the parent fullerenes.

  2. May the Best Molecule Win: Competition ESI Mass Spectrometry

    PubMed Central

    Laughlin, Sarah; Wilson, W. David

    2015-01-01

    Electrospray ionization mass spectrometry has become invaluable in the characterization of macromolecular biological systems such as nucleic acids and proteins. Recent advances in the field of mass spectrometry and the soft conditions characteristic of electrospray ionization allow for the investigation of non-covalent interactions among large biomolecules and ligands. Modulation of genetic processes through the use of small molecule inhibitors with the DNA minor groove is gaining attention as a potential therapeutic approach. In this review, we discuss the development of a competition method using electrospray ionization mass spectrometry to probe the interactions of multiple DNA sequences with libraries of minor groove binding molecules. Such an approach acts as a high-throughput screening method to determine important information including the stoichiometry, binding mode, cooperativity, and relative binding affinity. In addition to small molecule-DNA complexes, we highlight other applications in which competition mass spectrometry has been used. A competitive approach to simultaneously investigate complex interactions promises to be a powerful tool in the discovery of small molecule inhibitors with high specificity and for specific, important DNA sequences. PMID:26501262

  3. Desorption in Mass Spectrometry.

    PubMed

    Usmanov, Dilshadbek Tursunbayevich; Ninomiya, Satoshi; Chen, Lee Chuin; Saha, Subhrakanti; Mandal, Mridul Kanti; Sakai, Yuji; Takaishi, Rio; Habib, Ahsan; Hiraoka, Kenzo; Yoshimura, Kentaro; Takeda, Sen; Wada, Hiroshi; Nonami, Hiroshi

    2017-01-01

    In mass spectrometry, analytes must be released in the gas phase. There are two representative methods for the gasification of the condensed samples, i.e., ablation and desorption. While ablation is based on the explosion induced by the energy accumulated in the condensed matrix, desorption is a single molecular process taking place on the surface. In this paper, desorption methods for mass spectrometry developed in our laboratory: flash heating/rapid cooling, Leidenfrost phenomenon-assisted thermal desorption (LPTD), solid/solid friction, liquid/solid friction, electrospray droplet impact (EDI) ionization/desorption, and probe electrospray ionization (PESI), will be described. All the methods are concerned with the surface and interface phenomena. The concept of how to desorb less-volatility compounds from the surface will be discussed.

  4. Desorption in Mass Spectrometry

    PubMed Central

    Usmanov, Dilshadbek Tursunbayevich; Ninomiya, Satoshi; Chen, Lee Chuin; Saha, Subhrakanti; Mandal, Mridul Kanti; Sakai, Yuji; Takaishi, Rio; Habib, Ahsan; Hiraoka, Kenzo; Yoshimura, Kentaro; Takeda, Sen; Wada, Hiroshi; Nonami, Hiroshi

    2017-01-01

    In mass spectrometry, analytes must be released in the gas phase. There are two representative methods for the gasification of the condensed samples, i.e., ablation and desorption. While ablation is based on the explosion induced by the energy accumulated in the condensed matrix, desorption is a single molecular process taking place on the surface. In this paper, desorption methods for mass spectrometry developed in our laboratory: flash heating/rapid cooling, Leidenfrost phenomenon-assisted thermal desorption (LPTD), solid/solid friction, liquid/solid friction, electrospray droplet impact (EDI) ionization/desorption, and probe electrospray ionization (PESI), will be described. All the methods are concerned with the surface and interface phenomena. The concept of how to desorb less-volatility compounds from the surface will be discussed. PMID:28337398

  5. Protein-protein interactions of tandem affinity purified protein kinases from rice.

    PubMed

    Rohila, Jai S; Chen, Mei; Chen, Shuo; Chen, Johann; Cerny, Ronald L; Dardick, Christopher; Canlas, Patrick; Fujii, Hiroaki; Gribskov, Michael; Kanrar, Siddhartha; Knoflicek, Lucas; Stevenson, Becky; Xie, Mingtang; Xu, Xia; Zheng, Xianwu; Zhu, Jian-Kang; Ronald, Pamela; Fromm, Michael E

    2009-08-19

    Eighty-eight rice (Oryza sativa) cDNAs encoding rice leaf expressed protein kinases (PKs) were fused to a Tandem Affinity Purification tag (TAP-tag) and expressed in transgenic rice plants. The TAP-tagged PKs and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by tandem mass spectrometry. Forty-five TAP-tagged PKs were recovered in this study and thirteen of these were found to interact with other rice proteins with a high probability score. In vivo phosphorylated sites were found for three of the PKs. A comparison of the TAP-tagged data from a combined analysis of 129 TAP-tagged rice protein kinases with a concurrent screen using yeast two hybrid methods identified an evolutionarily new rice protein that interacts with the well conserved cell division cycle 2 (CDC2) protein complex.

  6. Protein-protein interactions of tandem affinity purification-tagged protein kinases in rice.

    PubMed

    Rohila, Jai S; Chen, Mei; Chen, Shuo; Chen, Johann; Cerny, Ronald; Dardick, Chris; Canlas, Patrick; Xu, Xia; Gribskov, Michael; Kanrar, Siddhartha; Zhu, Jian-Kang; Ronald, Pamela; Fromm, Michael E

    2006-04-01

    Forty-one rice cDNAs encoding protein kinases were fused to the tandem affinity purification (TAP) tag and expressed in transgenic rice plants. The TAP-tagged kinases and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by mass spectrometry. Ninety-five percent of the TAP-tagged kinases were recovered. Fifty-six percent of the TAP-tagged kinases were found to interact with other rice proteins. A number of these interactions were consistent with known protein complexes found in other species, validating the TAP-tag method in rice plants. Phosphorylation sites were identified on four of the kinases that interacted with either 14-3-3 proteins or cyclins.

  7. Protein-Protein Interactions of Tandem Affinity Purified Protein Kinases from Rice

    PubMed Central

    Rohila, Jai S.; Chen, Mei; Chen, Shuo; Chen, Johann; Cerny, Ronald L.; Dardick, Christopher; Canlas, Patrick; Fujii, Hiroaki; Gribskov, Michael; Kanrar, Siddhartha; Knoflicek, Lucas; Stevenson, Becky; Xie, Mingtang; Xu, Xia; Zheng, Xianwu; Zhu, Jian-Kang; Ronald, Pamela; Fromm, Michael E.

    2009-01-01

    Eighty-eight rice (Oryza sativa) cDNAs encoding rice leaf expressed protein kinases (PKs) were fused to a Tandem Affinity Purification tag (TAP-tag) and expressed in transgenic rice plants. The TAP-tagged PKs and interacting proteins were purified from the T1 progeny of the transgenic rice plants and identified by tandem mass spectrometry. Forty-five TAP-tagged PKs were recovered in this study and thirteen of these were found to interact with other rice proteins with a high probability score. In vivo phosphorylated sites were found for three of the PKs. A comparison of the TAP-tagged data from a combined analysis of 129 TAP-tagged rice protein kinases with a concurrent screen using yeast two hybrid methods identified an evolutionarily new rice protein that interacts with the well conserved cell division cycle 2 (CDC2) protein complex. PMID:19690613

  8. Identification of proteins associated with RNA polymerase III using a modified tandem chromatin affinity purification.

    PubMed

    Nguyen, Ngoc-Thuy-Trinh; Saguez, Cyril; Conesa, Christine; Lefebvre, Olivier; Acker, Joël

    2015-02-01

    To identify the proteins associated with the RNA polymerase III (Pol III) machinery in exponentially growing yeast cells, we developed our own tandem chromatin affinity purification procedure (TChAP) after in vivo cross-link, allowing a reproducible and good recovery of the protein bait and its associated partners. In contrast to TFIIIA that could only be purified as a free protein, this protocol allows us to capture free Pol III together with Pol III bound on its target genes. Transcription factors, elongation factors, RNA-associated proteins and proteins involved in Pol III biogenesis were identified by mass spectrometry. Interestingly, the presence of all the TFIIIB subunits found associated with Pol III together with the absence of TFIIIC and chromatin factors including histones suggest that DNA-bound Pol III purified using TChAP is mainly engaged in transcription reinitiation.

  9. Single-Step Affinity Purification of ERK Signaling Complexes Using the Streptavidin-Binding Peptide (SBP) Tag.

    PubMed

    Yang, Liu; Veraksa, Alexey

    2017-01-01

    Elucidation of biological functions of signaling proteins is facilitated by studying their protein-protein interaction networks. Affinity purification combined with mass spectrometry (AP-MS) has become a favorite method to study protein complexes. Here we describe a procedure for single-step purification of ERK (Rolled) and associated proteins from Drosophila cultured cells. The use of the streptavidin-binding peptide (SBP) tag allows for a highly efficient isolation of native ERK signaling complexes, which are suitable for subsequent analysis by mass spectrometry. Our analysis of the ERK interactome has identified both known and novel signaling components. This method can be easily adapted for SBP-based purification of protein complexes in any expression system.

  10. Affine connection form of Regge calculus

    NASA Astrophysics Data System (ADS)

    Khatsymovsky, V. M.

    2016-12-01

    Regge action is represented analogously to how the Palatini action for general relativity (GR) as some functional of the metric and a general connection as independent variables represents the Einstein-Hilbert action. The piecewise flat (or simplicial) spacetime of Regge calculus is equipped with some world coordinates and some piecewise affine metric which is completely defined by the set of edge lengths and the world coordinates of the vertices. The conjugate variables are the general nondegenerate matrices on the three-simplices which play the role of a general discrete connection. Our previous result on some representation of the Regge calculus action in terms of the local Euclidean (Minkowsky) frame vectors and orthogonal connection matrices as independent variables is somewhat modified for the considered case of the general linear group GL(4, R) of the connection matrices. As a result, we have some action invariant w.r.t. arbitrary change of coordinates of the vertices (and related GL(4, R) transformations in the four-simplices). Excluding GL(4, R) connection from this action via the equations of motion we have exactly the Regge action for the considered spacetime.

  11. [Separation of osteoclasts by lectin affinity chromatography].

    PubMed

    Itokazu, M; Tan, A; Tanaka, S

    1991-09-01

    Newborn rat calvaria bone cells obtained by digestion were fractionated on columns of wheat-germ agglutinin (WGA) sepharose 6MB for osteoclast isolation. The initial nonspecific binding cells which were passed through the WGA sepharose column by a buffer acquired a high enzyme activity of alkaline phosphatase, but not that of acid phosphatase. However, elution of cells using a buffer with the addition of N-acetyl-D-glucosamine resulted in a high acid phosphatase activity but no alkaline phosphatase activity. The former WGA binding negative fraction enriched osteoblasts averaging 30 microns in size. The latter WGA binding positive fraction enriched osteoclasts ranging from 20 microns to 60 microns in size. The electron-microscope clearly demonstrated the cellular details of osteoclasts. Isolated cell counts showed a ratio of six to four. These results indicate that our method of osteoclast isolation is simple and useful in lectin affinity chromatography because all cells have sugar moieties on their surface and the binding of osteoclasts can be reversed by the addition of specific lectin-binding sugars to the eluting buffer.

  12. Prostate Cancer and Bone: The Elective Affinities

    PubMed Central

    2014-01-01

    The onset of metastases dramatically changes the prognosis of prostate cancer patients, determining increased morbidity and a drastic fall in survival expectancy. Bone is a common site of metastases in few types of cancer, and it represents the most frequent metastatic site in prostate cancer. Of note, the prevalence of tumor relapse to the bone appears to be increasing over the years, likely due to a longer overall survival of prostate cancer patients. Bone tropism represents an intriguing challenge for researchers also because the preference of prostate cancer cells for the bone is the result of a sequential series of targetable molecular events. Many factors have been associated with the peculiar ability of prostate cancer cells to migrate in bone marrow and to determine mixed osteoblastic/osteolytic lesions. As anticipated by the success of current targeted therapy aimed to block bone resorption, a better understanding of molecular affinity between prostate cancer and bone microenvironment will permit us to cure bone metastasis and to improve prognosis of prostate cancer patients. PMID:24971315

  13. Multiplexed protein profiling by sequential affinity capture

    PubMed Central

    Ayoglu, Burcu; Birgersson, Elin; Mezger, Anja; Nilsson, Mats; Uhlén, Mathias; Nilsson, Peter

    2016-01-01

    Antibody microarrays enable parallelized and miniaturized analysis of clinical samples, and have proven to provide novel insights for the analysis of different proteomes. However, there are concerns that the performance of such direct labeling and single antibody assays are prone to off‐target binding due to the sample context. To improve selectivity and sensitivity while maintaining the possibility to conduct multiplexed protein profiling, we developed a multiplexed and semi‐automated sequential capture assay. This novel bead‐based procedure encompasses a first antigen capture, labeling of captured protein targets on magnetic particles, combinatorial target elution and a read‐out by a secondary capture bead array. We demonstrate in a proof‐of‐concept setting that target detection via two sequential affinity interactions reduced off‐target contribution, while lowered background and noise levels, improved correlation to clinical values compared to single binder assays. We also compared sensitivity levels with single binder and classical sandwich assays, explored the possibility for DNA‐based signal amplification, and demonstrate the applicability of the dual capture bead‐based antibody microarray for biomarker analysis. Hence, the described concept enhances the possibilities for antibody array assays to be utilized for protein profiling in body fluids and beyond. PMID:26935855

  14. Cambrian trilobites with Siberian affinities, southwestern Alaska

    SciTech Connect

    Palmer, A.R.; Egbert, R.M.; Sullivan, R.; Knoth, J.S.

    1985-02-01

    Cambrian trilobites occur in two levels (about 7 m apart) in the core of a large, complex anticlinal structure in the area between the Taylor Mountains and the Hoholitna River in southwestern Alaska. The lower collection contains Erbia, Macannaia (a species close to Soviet forms described as Pagetia ferox Lermontova), two species of Kootenia (including one perhaps cospecific with forms from the central Brooks range), and several species of ptychoparioid trilobites. It is clear that biogeographic affinities are with the transitional facies of the eastern Siberian platform and the south Siberian foldbelt. In Soviet terms, the age of the collection falls in a disputed interval called latest Early Cambrian (Tojonian) by some authors, and earliest Middle Cambrian (Amgan) by others. In North American terms, Macannaia is known only from early Middle Cambrian beds. The younger collection contains abundant agnostids, a variety of conocoryphids, Paradoxides, and several species of ptychoparioid trilobites. This is an assemblage of undoubted late Middle Cambrian age, comparable to faunas described from the Maya State of the Siberian platform and the Paradoxides paradoxissimus Stage of the Baltic region. Both faunas are from ocean-facing or outer shelf environments. None of the key non-agnostid or non-pagetiid elements have been seen previously in deposits of Cambrian North America.

  15. Affinity of guanosine derivatives for polycytidylate revisited

    NASA Technical Reports Server (NTRS)

    Kanavarioti, A.; Hurley, T. B.; Baird, E. E.

    1995-01-01

    Evidence is presented for complexation of guanosine 5'-monophosphate 2-methylimidazolide (2-MeImpG) with polycytidylate (poly(C)) at pH 8.0 and 23 degrees C in the presence of 1.0 M NaCl2 and 0.2 M MgCl2 in water. The association of 2-MeImpG with poly(C) was investigated using UV-vis spectroscopy as well as by monitoring the kinetics of the nucleophilic substitution reaction of the imidazole moiety by amines. The results of both methods are consistent with moderately strong poly(C) 2-MeImpG complexation and the spectrophotometric measurements allowed the construction of a binding isotherm with a concentration of 2-MeImpG equal to 5.55 +/- 0.15 mM at half occupancy. UV spectroscopy was employed to establish the binding of other guanosine derivatives on poly(C). These derivatives are guanosine 5'-monophosphate (5'GMP), guanosine 5'-monophosphate imidazolide (ImpG), and guanosine 5'-monophosphate morpholidate (morpG). Within experimental error these guanosine derivatives exhibit the same affinity for poly(C) as 2-MeImpG.

  16. Macroporous chitin affinity membranes for lysozyme separation.

    PubMed

    Ruckenstein, E; Zeng, X

    1997-12-20

    Macroporous chitin membranes with high, controlled porosity and good mechanical properties have been prepared using a technique developed in this laboratory based on silica particles as porogen. They were employed for the affinity separation of lysozyme. Chitin membranes (1 mm thickness) can be operated at high fluxes (>/=1.1 mL/min/cm(2)) corresponding to pressure drops >/=2 psi. Their adsorption capacity for lysozyme ( approximately 50 mg/mL membrane) is by an order of magnitude higher than that of the chitin beads employed in column separation. In a binary mixture of lysozyme and ovalbumin, the membranes showed very high selectivity towards lysozyme. The effect of some important operation parameters, such as the flow rates during loading and elution were investigated. Lysozyme of very high purity (>98%) was obtained from a mixture of lysozyme and ovalbumin, and from egg white. The results indicate that the macroporous chitin membranes can be used for the separation, purification, and recovery of lysozyme at large scale. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 610-617, 1997.

  17. Influence of Sulfolane on ESI-MS Measurements of Protein-Ligand Affinities

    NASA Astrophysics Data System (ADS)

    Yao, Yuyu; Richards, Michele R.; Kitova, Elena N.; Klassen, John S.

    2016-03-01

    The results of an investigation into the influence of sulfolane, a commonly used supercharging agent, on electrospray ionization mass spectrometry (ESI-MS) measurements of protein-ligand affinities are described. Binding measurements carried out on four protein-carbohydrate complexes, lysozyme with β- d-GlcNAc-(1→4)-β- d-GlcNAc-(1→4)-β- d-GlcNAc-(1→4)- d-GlcNAc, a single chain variable fragment and α- d-Gal-(1→2)-[α- d-Abe-(1→3)]-α- d-Man-OCH3, cholera toxin B subunit homopentamer with β- d-Gal-(1→3)-β- d-GalNAc-(1→4)[α- d-Neu5Ac-(2→3)]-β- d-Gal-(1→4)-β- d-Glc, and a fragment of galectin 3 and α- l-Fuc-(1→2)-β- d-Gal-(1→3)-β- d-GlcNAc-(1→3)-β- d-Gal-(1→4)-β- d-Glc, revealed that sulfolane generally reduces the apparent (as measured by ESI-MS) protein-ligand affinities. To establish the origin of this effect, a detailed study was undertaken using the lysozyme-tetrasaccharide interaction as a model system. Measurements carried out using isothermal titration calorimetry (ITC), circular dichroism, and nuclear magnetic resonance spectroscopies reveal that sulfolane reduces the binding affinity in solution but does not cause any significant change in the higher order structure of lysozyme or to the intermolecular interactions. These observations confirm that changes to the structure of lysozyme in bulk solution are not responsible for the supercharging effect induced by sulfolane. Moreover, the agreement between the ESI-MS and ITC-derived affinities indicates that there is no dissociation of the complex during ESI or in the gas phase (i.e., in-source dissociation). This finding suggests that supercharging of lysozyme by sulfolane is not related to protein unfolding during the ESI process. Binding measurements performed using liquid sample desorption ESI-MS revealed that protein supercharging with sulfolane can be achieved without a reduction in affinity.

  18. Germinal center reaction: antigen affinity and presentation explain it all.

    PubMed

    Oropallo, Michael A; Cerutti, Andrea

    2014-07-01

    The selection and expansion of B cells undergoing affinity maturation in the germinal center is a hallmark of humoral immunity. A recent paper in Nature provides new insights into the relationships between the affinity of the immunoglobulin receptor for antigen, the ability of B cells to present antigen to T cells, and the processes of selection, mutation, and clonal expansion in the germinal center.

  19. Striving for Empathy: Affinities, Alliances and Peer Sexuality Educators

    ERIC Educational Resources Information Center

    Fields, Jessica; Copp, Martha

    2015-01-01

    Peer sexuality educators' accounts of their work reveal two approaches to empathy with their students: affinity and alliance. "Affinity-based empathy" rests on the idea that the more commonalities sexuality educators and students share (or perceive they share), the more they will be able to empathise with one another, while…

  20. Conformational kinetics reveals affinities of protein conformational states.

    PubMed

    Daniels, Kyle G; Suo, Yang; Oas, Terrence G

    2015-07-28

    Most biological reactions rely on interplay between binding and changes in both macromolecular structure and dynamics. Practical understanding of this interplay requires detection of critical intermediates and determination of their binding and conformational characteristics. However, many of these species are only transiently present and they have often been overlooked in mechanistic studies of reactions that couple binding to conformational change. We monitored the kinetics of ligand-induced conformational changes in a small protein using six different ligands. We analyzed the kinetic data to simultaneously determine both binding affinities for the conformational states and the rate constants of conformational change. The approach we used is sufficiently robust to determine the affinities of three conformational states and detect even modest differences in the protein's affinities for relatively similar ligands. Ligand binding favors higher-affinity conformational states by increasing forward conformational rate constants and/or decreasing reverse conformational rate constants. The amounts by which forward rate constants increase and reverse rate constants decrease are proportional to the ratio of affinities of the conformational states. We also show that both the affinity ratio and another parameter, which quantifies the changes in conformational rate constants upon ligand binding, are strong determinants of the mechanism (conformational selection and/or induced fit) of molecular recognition. Our results highlight the utility of analyzing the kinetics of conformational changes to determine affinities that cannot be determined from equilibrium experiments. Most importantly, they demonstrate an inextricable link between conformational dynamics and the binding affinities of conformational states.

  1. Affine group formulation of the Standard Model coupled to gravity

    SciTech Connect

    Chou, Ching-Yi; Ita, Eyo; Soo, Chopin

    2014-04-15

    In this work we apply the affine group formalism for four dimensional gravity of Lorentzian signature, which is based on Klauder’s affine algebraic program, to the formulation of the Hamiltonian constraint of the interaction of matter and all forces, including gravity with non-vanishing cosmological constant Λ, as an affine Lie algebra. We use the hermitian action of fermions coupled to gravitation and Yang–Mills theory to find the density weight one fermionic super-Hamiltonian constraint. This term, combined with the Yang–Mills and Higgs energy densities, are composed with York’s integrated time functional. The result, when combined with the imaginary part of the Chern–Simons functional Q, forms the affine commutation relation with the volume element V(x). Affine algebraic quantization of gravitation and matter on equal footing implies a fundamental uncertainty relation which is predicated upon a non-vanishing cosmological constant. -- Highlights: •Wheeler–DeWitt equation (WDW) quantized as affine algebra, realizing Klauder’s program. •WDW formulated for interaction of matter and all forces, including gravity, as affine algebra. •WDW features Hermitian generators in spite of fermionic content: Standard Model addressed. •Constructed a family of physical states for the full, coupled theory via affine coherent states. •Fundamental uncertainty relation, predicated on non-vanishing cosmological constant.

  2. Tending to Change: Toward a Situated Model of Affinity Spaces

    ERIC Educational Resources Information Center

    Bommarito, Dan

    2014-01-01

    The concept of affinity spaces, a theoretical construct used to analyze literate activity from a spatial perspective, has gained popularity among scholars of literacy studies and, particularly, video-game studies. This article seeks to expand current notions of affinity spaces by identifying key assumptions that have limited researchers'…

  3. Hybrid instruments for mass spectrometry/mass spectrometry

    SciTech Connect

    Glish, G.L.; McLuckey, S.A.

    1986-01-01

    In order to refine further the technique of mass spectrometry/mass spectrometry efforts are being made to combine the desirable features of sector based tandem instruments with those of triple quadrupole mass spectrometers. This has resulted in the construction of tandem mass spectrometers which incorporate both sector type analyzers and quadrupole mass filters. These so-called hybrid instruments, designed specifically for mass spectrometry/mass spectrometry applications, are appearing in a variety of geometries each with unique features. This review describes the hybrid instruments reported to data and discusses general considerations for evaluating hybrid instruments with regard to application. 100 references.

  4. Affinity based information diffusion model in social networks

    NASA Astrophysics Data System (ADS)

    Liu, Hongli; Xie, Yun; Hu, Haibo; Chen, Zhigao

    2014-12-01

    There is a widespread intuitive sense that people prefer participating in spreading the information in which they are interested. The affinity of people with information disseminated can affect the information propagation in social networks. In this paper, we propose an information diffusion model incorporating the mechanism of affinity of people with information which considers the fitness of affinity values of people with affinity threshold of the information. We find that the final size of information diffusion is affected by affinity threshold of the information, average degree of the network and the probability of people's losing their interest in the information. We also explore the effects of other factors on information spreading by numerical simulations and find that the probabilities of people's questioning and confirming the information can affect the propagation speed, but not the final scope.

  5. Camelid VHH affinity ligands enable separation of closely related biopharmaceuticals

    PubMed Central

    Pabst, Timothy M.; Wendeler, Michaela; Wang, Xiangyang; Bezemer, Sandra; Hermans, Pim

    2016-01-01

    Abstract Interest in new and diverse classes of molecules such as recombinant toxins, enzymes, and blood factors continues to grow for use a biotherapeutics. Compared to monoclonal antibodies, these novel drugs typically lack a commercially available affinity chromatography option, which leads to greater process complexity, longer development timelines, and poor platformability. To date, for both monoclonal antibodies and novel molecules, affinity chromatography has been mostly reserved for separation of process‐related impurities such as host cell proteins and DNA. Reports of affinity purification of closely related product variants and modified forms are much rarer. In this work we describe custom affinity chromatography development using camelid VHH antibody fragments as "tunable" immunoaffinity ligands for separation of product‐related impurities. One example demonstrates high selectivity for a recombinant immunotoxin where no binding was observed for an undesired deamidated species. Also discussed is affinity purification of a coagulation factor through specific recognition of the gamma‐carboxylglutamic acid domain. PMID:27677057

  6. Affinity Monolith-Integrated Microchips for Protein Purification and Concentration.

    PubMed

    Gao, Changlu; Sun, Xiuhua; Wang, Huaixin; Qiao, Wei; Hu, Bo

    2016-01-01

    Affinity chromatography is a valuable method to purify and concentrate minute amount of proteins. Monoliths with epoxy groups for affinity immobilization were prepared by direct in-situ photopolymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in porogenic solvents consisting of 1-dodecanol and cyclohexanol. By integrating affinity monoliths onto a microfluidic system, targeted biomolecules can be captured and retained on affinity column, while other biomolecules having no specific interactions toward the immobilized ligands flow through the microchannel. Therefore, proteins which remain on the affinity column are purified and concentrated, and then eluted by appropriate solutions and finally, separated by microchip capillary electrophoresis. This integrated microfluidic device has been applied to the purification and separation of specific proteins (FITC-labeled human serum albumin and IgG) in a mixture.

  7. Optimal T-cell receptor affinity for inducing autoimmunity

    PubMed Central

    Koehli, Sabrina; Naeher, Dieter; Galati-Fournier, Virginie; Zehn, Dietmar; Palmer, Ed

    2014-01-01

    T-cell receptor affinity for self-antigen has an important role in establishing self-tolerance. Three transgenic mouse strains expressing antigens of variable affinity for the OVA transgenic-I T-cell receptor were generated to address how TCR affinity affects the efficiency of negative selection, the ability to prime an autoimmune response, and the elimination of the relevant target cell. Mice expressing antigens with an affinity just above the negative selection threshold exhibited the highest risk of developing experimental autoimmune diabetes. The data demonstrate that close to the affinity threshold for negative selection, sufficient numbers of self-reactive T cells escape deletion and create an increased risk for the development of autoimmunity. PMID:25411315

  8. Affinity Regulates Spatial Range of EGF Receptor Autocrine Ligand Binding

    SciTech Connect

    Dewitt, Ann; Iida, Tomoko; Lam, Ho-Yan; Hill, Virginia; Wiley, H S.; Lauffenburger, Douglas A.

    2002-08-08

    Proper spatial localization of EGFR signaling activated by autocrine ligands represents a critical factor in embryonic development as well as tissue organization and function, and ligand/receptor binding affinity is among the molecular and cellular properties suggested to play a role in governing this localization. The authors employ a computational model to predict how receptor-binding affinity affects local capture of autocrine ligand vis-a-vis escape to distal regions, and provide experimental test by constructing cell lines expressing EGFR along with either wild-type EGF or a low-affinity mutant, EGF{sup L47M}. The model predicts local capture of a lower affinity autocrine ligand to be less efficient when the ligand production rate is small relative to receptor appearance rate. The experimental data confirm this prediction, demonstrating that cells can use ligand/receptor binding affinity to regulate ligand spatial distribution when autocrine ligand production is limiting for receptor signaling.

  9. Chasing polys: Interdisciplinary affinity and its connection to physics identity

    NASA Astrophysics Data System (ADS)

    Scott, Tyler D.

    This research is based on two motivations that merge by means of the frameworks of interdisciplinary affinity and physics identity. First, a goal of education is to develop interdisciplinary abilities in students' thinking and work. But an often ignored factor is students interests and beliefs about being interdisciplinary. Thus, this work develops and uses a framework called interdisciplinary affinity. It encompasses students interests in making connections across disciplines and their beliefs about their abilities to make those connections. The second motivation of this research is to better understand how to engage more students with physics. Physics identity describes how a student sees themselves in relation to physics. By understanding how physics identity is developed, researchers and educators can identify factors that increase interest and engagement in physics classrooms. Therefore, physics identity was used in conjunction with interdisciplinary affinity. Using a mixed methods approach, this research used quantitative data to identify the relationships interdisciplinary affinity has with physics identity and the physics classroom. These connections were explored in more detail using a case study of three students in a high school physics class. Results showed significant and positive relationships between interdisciplinary affinity and physics identity, including the individual interest and recognition components of identity. It also identified characteristics of physics classrooms that had a significant, positive relationship with interdisciplinary affinity. The qualitative case study highlighted the importance of student interest to the relationship between interdisciplinary affinity and physics identity. It also identified interest and mastery orientation as key to understanding the link between interdisciplinary affinity and the physics classroom. These results are a positive sign that by understanding interdisciplinary affinity and physics identity

  10. Affinity capillary electrophoresis: the theory of electromigration.

    PubMed

    Dubský, Pavel; Dvořák, Martin; Ansorge, Martin

    2016-12-01

    We focus on the state-of-the-art theory of electromigration under single and multiple complexation equilibrium. Only 1:1 complexation stoichiometry is discussed because of its unique status in the field of affinity capillary electrophoresis (ACE). First, we summarize the formulas for the effective mobility in various ACE systems as they appeared since the pioneering days in 1992 up to the most recent theories till 2015. Disturbing phenomena that do not alter the mobility of the analyte directly but cause an unexpected peak broadening have been studied only recently and are also discussed in this paper. Second, we turn our attention to the viscosity effects in ACE. Change in the background electrolyte viscosity is unavoidable in ACE but numerous observations scattered throughout the literature have not been reviewed previously. This leads to an uncritical employment of correction factors that may or may not be appropriate in practice. Finally, we consider the ionic strength effects in ACE, too. Limitations of the current theories are also discussed and the tasks identified where open problems still prevail. Graphical Abstract A weak base (A) undergoes an acidic-basic equilibria (in blue) and migrates with an electrophoretic mobility of [Formula: see text]. Simultaneously, it interacts with a selector (sel) while the analyte-selector complex migrates with an electrophoretic mobility of [Formula: see text]. The strength of the interaction (in orange) is governed by the binding constant, K A , and the concentration of the selector, c sel . This all gives the analyte an effective mobility of [Formula: see text] and moves it out of the zero position (EOF; right top insert). The interaction of the positively charged analyte with the neutral selector slows down the analyte with increasing selector concentration (right bottom insert).

  11. Targeting Protein-Protein Interactions with Trimeric Ligands: High Affinity Inhibitors of the MAGUK Protein Family

    PubMed Central

    Nissen, Klaus B.; Haugaard-Kedström, Linda M.; Wilbek, Theis S.; Nielsen, Line S.; Åberg, Emma; Kristensen, Anders S.; Bach, Anders; Jemth, Per; Strømgaard, Kristian

    2015-01-01

    PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins. PMID:25658767

  12. Targeting protein-protein interactions with trimeric ligands: high affinity inhibitors of the MAGUK protein family.

    PubMed

    Nissen, Klaus B; Haugaard-Kedström, Linda M; Wilbek, Theis S; Nielsen, Line S; Åberg, Emma; Kristensen, Anders S; Bach, Anders; Jemth, Per; Strømgaard, Kristian

    2015-01-01

    PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins.

  13. Combustion diagnostics by laser spectrometry

    NASA Astrophysics Data System (ADS)

    Kitagawa, Kuniyuki; Morita, Shigeaki; Kodama, Kenji; Matsumoto, Kozo

    2009-03-01

    We have developed three different types of visualization methods for energy conversion systems by means of laser spectrometry. (1) Laser-induced fluorescence (LIF) spectroscopy and (2) laser ionization mass spectrometry (LIMS) have been applied to visualization of chemical species in combustion fields of flames. (3) Near-infrared laser absorption spectroscopy has been used for visualization of water in a polymer electrolyte fuel cell (PEFC). Complex physicochemical processes in the energy conversion systems have been revealed by laser spectrometry.

  14. Aggregation of two carboxylic derivatives of porphyrin and their affinity to bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Yin, Yao-Bing; Wang, Yi-Nong; Ma, Jian-Biao

    2006-07-01

    Aggregation of two porphyrin derivatives with carboxylic groups, 4-oxo-4-((4-(10,15,20-triphenyl-21 H,23 H-porphin-5-yl)phenyl)amino)butanoic acid (MAC) and 4,4',4″,4‴-[21 H,23 H-porphine-5,10,15,20-tetrayltetrakis(4,1-phenyleneimino)]tetrakis(4-oxo-butanoic acid) (TA4C), and their affinity to bovine serum albumin were investigated via absorption spectrometry, 1H NMR and fluorescence spectrometry. MAC and its complexes with β-cyclodextrin could form aggregates in an aqueous solution while TA4C was self-associated loosely. From the absorbance profiles of MAC in the titration of bovine serum albumin, hypochromicity was observed without any shift of the maximum absorbance wavelength. In both absorption spectra of TA4C in aqueous solutions and in solid state, three Q bands appeared in the visible region. In the measurements of absorption and fluorescence spectra upon titration of BSA, some spectral changes of TA4C were observed. The whole procedure of titration could be divided into three successive stages. The three-banded profiles of TA4C might be explained according to a loose dimer model.

  15. Aggregation of two carboxylic derivatives of porphyrin and their affinity to bovine serum albumin.

    PubMed

    Yin, Yao-Bing; Wang, Yi-Nong; Ma, Jian-Biao

    2006-07-01

    Aggregation of two porphyrin derivatives with carboxylic groups, 4-oxo-4-((4-(10,15,20-triphenyl-21H,23H-porphin-5-yl)phenyl)amino)butanoic acid (MAC) and 4,4',4'',4'''-[21H,23H-porphine-5,10,15,20-tetrayltetrakis(4,1-phenyleneimino)]tetrakis(4-oxo-butanoic acid) (TA4C), and their affinity to bovine serum albumin were investigated via absorption spectrometry, (1)H NMR and fluorescence spectrometry. MAC and its complexes with beta-cyclodextrin could form aggregates in an aqueous solution while TA4C was self-associated loosely. From the absorbance profiles of MAC in the titration of bovine serum albumin, hypochromicity was observed without any shift of the maximum absorbance wavelength. In both absorption spectra of TA4C in aqueous solutions and in solid state, three Q bands appeared in the visible region. In the measurements of absorption and fluorescence spectra upon titration of BSA, some spectral changes of TA4C were observed. The whole procedure of titration could be divided into three successive stages. The three-banded profiles of TA4C might be explained according to a loose dimer model.

  16. Magnetic Microbead Affinity Selection Screening (MagMASS) of Botanical Extracts for Inhibitors of 15-Lipoxygenase.

    PubMed

    Rush, Michael D; Walker, Elisabeth M; Burton, Tristesse; van Breemen, Richard B

    2016-11-23

    To expedite the identification of active natural products in complex mixtures such as botanical extracts, a magnetic microbead affinity selection screening (MagMASS) procedure was developed. This technique utilizes target proteins immobilized on magnetic beads for rapid bioaffinity isolation of ligands from complex mixtures. A MagMASS method was developed and validated for 15-lipoxygenase. As a proof of concept, several North American prairie plants used medicinally by Native Americans were extracted with MeOH and screened. A hit from an extract of Proserpinaca palustris, also known as mermaid weed, was flagged for further characterization using high-resolution tandem mass spectrometry, dereplication, and identification using XCMS online. Through the application of high-resolution product ion tandem mass spectrometry, comparison with natural product databases, and confirmation using standards, the hit was identified as quercitrin, which is a known inhibitor of 15-lipoxygenase. The overall workflow of MagMASS is faster and more amendable to automation than alternative methods designed for screening botanical extracts or complex mixtures of combinatorial libraries.

  17. Enantioselectivity of mass spectrometry: challenges and promises.

    PubMed

    Awad, Hanan; El-Aneed, Anas

    2013-01-01

    With the fast growing market of pure enantiomer drugs and bioactive molecules, new chiral-selective analytical tools have been instigated including the use of mass spectrometry (MS). Even though MS is one of the best analytical tools that has efficiently been used in several pharmaceutical and biological applications, traditionally MS is considered as a "chiral-blind" technique. This limitation is due to the MS inability to differentiate between two enantiomers of a chiral molecule based merely on their masses. Several approaches have been explored to assess the potential role of MS in chiral analysis. The first approach depends on the use of MS-hyphenated techniques utilizing fast and sensitive chiral separation tools such as liquid chromatography (LC), gas chromatography (GC), and capillary electrophoresis (CE) coupled to MS detector. More recently, several alternative separation techniques have been evaluated such as supercritical fluid chromatography (SFC) and capillary electrochromatography (CEC); the latter being a hybrid technique that combines the efficiency of CE with the selectivity of LC. The second approach is based on using the MS instrument solely for the chiral recognition. This method depends on the behavioral differences between enantiomers towards a foreign molecule and the ability of MS to monitor such differences. These behavioral differences can be divided into three types: (i) differences in the enantiomeric affinity for association with the chiral selector, (ii) differences of the enantiomeric exchange rate with a foreign reagent, and (iii) differences in the complex MS dissociation behaviors of the enantiomers. Most recently, ion mobility spectrometry was introduced to qualitatively and quantitatively evaluate chiral compounds. This article provides an overview of MS role in chiral analysis by discussing MS based methodologies and presenting the challenges and promises associated with each approach.

  18. Analysis of biomolecular interactions using affinity microcolumns: A review

    PubMed Central

    Zheng, Xiwei; Li, Zhao; Beeram, Sandya; Podariu, Maria; Matsuda, Ryan; Pfaunmiller, Erika L.; White, Christopher J.; Carter, NaTasha; Hage, David S.

    2014-01-01

    Affinity chromatography has become an important tool for characterizing biomolecular interactions. The use of affinity microcolumns, which contain immobilized binding agents and have volumes in the mid-to-low microliter range, has received particular attention in recent years. Potential advantages of affinity microcolumns include the many analysis and detection formats that can be used with these columns, as well as the need for only small amounts of supports and immobilized binding agents. This review examines how affinity microcolumns have been used to examine biomolecular interactions. Both capillary-based microcolumns and short microcolumns are considered. The use of affinity microcolumns with zonal elution and frontal analysis methods are discussed. The techniques of peak decay analysis, ultrafast affinity extraction, split-peak analysis, and band-broadening studies are also explored. The principles of these methods are examined and various applications are provided to illustrate the use of these methods with affinity microcolumns. It is shown how these techniques can be utilized to provide information on the binding strength and kinetics of an interaction, as well as on the number and types of binding sites. It is further demonstrated how information on competition or displacement effects can be obtained by these methods. PMID:24572459

  19. "Magic" Ionization Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Trimpin, Sarah

    2016-01-01

    The systematic study of the temperature and pressure dependence of matrix-assisted ionization (MAI) led us to the discovery of the seemingly impossible, initially explained by some reviewers as either sleight of hand or the misinterpretation by an overzealous young scientist of results reported many years before and having little utility. The "magic" that we were attempting to report was that with matrix assistance, molecules, at least as large as bovine serum albumin (66 kDa), are lifted into the gas phase as multiply charged ions simply by exposure of the matrix:analyte sample to the vacuum of a mass spectrometer. Applied heat, a laser, or voltages are not necessary to achieve charge states and ion abundances only previously observed with electrospray ionization (ESI). The fundamentals of how solid phase volatile or nonvolatile compounds are converted to gas-phase ions without added energy currently involves speculation providing a great opportunity to rethink mechanistic understanding of ionization processes used in mass spectrometry. Improved understanding of the mechanism(s) of these processes and their connection to ESI and matrix-assisted laser desorption/ionization may provide opportunities to further develop new ionization strategies for traditional and yet unforeseen applications of mass spectrometry. This Critical Insights article covers developments leading to the discovery of a seemingly magic ionization process that is simple to use, fast, sensitive, robust, and can be directly applied to surface characterization using portable or high performance mass spectrometers.

  20. "Magic" Ionization Mass Spectrometry.

    PubMed

    Trimpin, Sarah

    2016-01-01

    The systematic study of the temperature and pressure dependence of matrix-assisted ionization (MAI) led us to the discovery of the seemingly impossible, initially explained by some reviewers as either sleight of hand or the misinterpretation by an overzealous young scientist of results reported many years before and having little utility. The “magic” that we were attempting to report was that with matrix assistance, molecules, at least as large as bovine serum albumin (66 kDa), are lifted into the gas phase as multiply charged ions simply by exposure of the matrix:analyte sample to the vacuum of a mass spectrometer. Applied heat, a laser, or voltages are not necessary to achieve charge states and ion abundances only previously observed with electrospray ionization (ESI). The fundamentals of how solid phase volatile or nonvolatile compounds are converted to gas-phase ions without added energy currently involves speculation providing a great opportunity to rethink mechanistic understanding of ionization processes used in mass spectrometry. Improved understanding of the mechanism(s) of these processes and their connection to ESI and matrix-assisted laser desorption/ionization may provide opportunities to further develop new ionization strategies for traditional and yet unforeseen applications of mass spectrometry. This Critical Insights article covers developments leading to the discovery of a seemingly magic ionization process that is simple to use, fast, sensitive, robust, and can be directly applied to surface characterization using portable or high performance mass spectrometers.

  1. Affinity+: Semi-Structured Brainstorming on Large Displays

    SciTech Connect

    Burtner, Edwin R.; May, Richard A.; Scarberry, Randall E.; LaMothe, Ryan R.; Endert, Alexander

    2013-04-27

    Affinity diagraming is a powerful method for encouraging and capturing lateral thinking in a group environment. The Affinity+ Concept was designed to improve the collaborative brainstorm process through the use of large display surfaces in conjunction with mobile devices like smart phones and tablets. The system works by capturing the ideas digitally and allowing users to sort and group them on a large touch screen manually. Additionally, Affinity+ incorporates theme detection, topic clustering, and other processing algorithms that help bring structured analytic techniques to the process without requiring explicit leadership roles and other overhead typically involved in these activities.

  2. Binding Affinities Controlled by Shifting Conformational Equilibria: Opportunities and Limitations

    PubMed Central

    Michielssens, Servaas; de Groot, Bert L.; Grubmüller, Helmut

    2015-01-01

    Conformational selection is an established mechanism in molecular recognition. Despite its power to explain binding events, it is hardly used in protein/ligand design to modulate molecular recognition. Here, we explore the opportunities and limitations of design by conformational selection. Using appropriate thermodynamic cycles, our approach predicts the effects of a conformational shift on binding affinity and also allows one to disentangle the effects induced by a conformational shift from other effects influencing the binding affinity. The method is assessed and applied to explain the contribution of a conformational shift on the binding affinity of six ubiquitin mutants showing different conformational shifts in six different complexes. PMID:25992736

  3. Characterization of the human submandibular/sublingual saliva glycoproteome using lectin affinity chromatography coupled to multidimensional protein identification technology.

    PubMed

    Gonzalez-Begne, Mireya; Lu, Bingwen; Liao, Lujian; Xu, Tao; Bedi, Gurrinder; Melvin, James E; Yates, John R

    2011-11-04

    In-depth analysis of the salivary proteome is fundamental to understanding the functions of salivary proteins in the oral cavity and to reveal disease biomarkers involved in different pathophysiological conditions, with the ultimate goal of improving patient diagnosis and prognosis. Submandibular and sublingual glands contribute saliva rich in glycoproteins to the total saliva output, making them valuable sources for glycoproteomic analysis. Lectin-affinity chromatography coupled to mass spectrometry-based shotgun proteomics was used to explore the submandibular/sublingual (SM/SL) saliva glycoproteome. A total of 262 N- and O-linked glycoproteins were identified by multidimensional protein identification technology (MudPIT). Only 38 were previously described in SM and SL salivas from the human salivary N-linked glycoproteome, while 224 were unique. Further comparison analysis with SM/SL saliva of the human saliva proteome, revealed 125 glycoproteins not formerly reported in this secretion. KEGG pathway analyses demonstrated that many of these glycoproteins are involved in processes such as complement and coagulation cascades, cell communication, glycosphingolipid biosynthesis neo-lactoseries, O-glycan biosynthesis, glycan structures-biosynthesis 2, starch and sucrose metabolism, peptidoglycan biosynthesis or others pathways. In summary, lectin-affinity chromatography coupled to MudPIT mass spectrometry identified many novel glycoproteins in SM/SL saliva. These new additions to the salivary proteome may prove to be a critical step for providing reliable biomarkers in the diagnosis of a myriad of oral and systemic diseases.

  4. Bidirectional elastic image registration using B-spline affine transformation.

    PubMed

    Gu, Suicheng; Meng, Xin; Sciurba, Frank C; Ma, Hongxia; Leader, Joseph; Kaminski, Naftali; Gur, David; Pu, Jiantao

    2014-06-01

    A registration scheme termed as B-spline affine transformation (BSAT) is presented in this study to elastically align two images. We define an affine transformation instead of the traditional translation at each control point. Mathematically, BSAT is a generalized form of the affine transformation and the traditional B-spline transformation (BST). In order to improve the performance of the iterative closest point (ICP) method in registering two homologous shapes but with large deformation, a bidirectional instead of the traditional unidirectional objective/cost function is proposed. In implementation, the objective function is formulated as a sparse linear equation problem, and a sub-division strategy is used to achieve a reasonable efficiency in registration. The performance of the developed scheme was assessed using both two-dimensional (2D) synthesized dataset and three-dimensional (3D) volumetric computed tomography (CT) data. Our experiments showed that the proposed B-spline affine model could obtain reasonable registration accuracy.

  5. Bidirectional Elastic Image Registration Using B-Spline Affine Transformation

    PubMed Central

    Gu, Suicheng; Meng, Xin; Sciurba, Frank C.; Wang, Chen; Kaminski, Naftali; Pu, Jiantao

    2014-01-01

    A registration scheme termed as B-spline affine transformation (BSAT) is presented in this study to elastically align two images. We define an affine transformation instead of the traditional translation at each control point. Mathematically, BSAT is a generalized form of the affine transformation and the traditional B-Spline transformation (BST). In order to improve the performance of the iterative closest point (ICP) method in registering two homologous shapes but with large deformation, a bi-directional instead of the traditional unidirectional objective / cost function is proposed. In implementation, the objective function is formulated as a sparse linear equation problem, and a sub-division strategy is used to achieve a reasonable efficiency in registration. The performance of the developed scheme was assessed using both two-dimensional (2D) synthesized dataset and three-dimensional (3D) volumetric computed tomography (CT) data. Our experiments showed that the proposed B-spline affine model could obtain reasonable registration accuracy. PMID:24530210

  6. A thermodynamic approach to the affinity optimization of drug candidates.

    PubMed

    Freire, Ernesto

    2009-11-01

    High throughput screening and other techniques commonly used to identify lead candidates for drug development usually yield compounds with binding affinities to their intended targets in the mid-micromolar range. The affinity of these molecules needs to be improved by several orders of magnitude before they become viable drug candidates. Traditionally, this task has been accomplished by establishing structure activity relationships to guide chemical modifications and improve the binding affinity of the compounds. As the binding affinity is a function of two quantities, the binding enthalpy and the binding entropy, it is evident that a more efficient optimization would be accomplished if both quantities were considered and improved simultaneously. Here, an optimization algorithm based upon enthalpic and entropic information generated by Isothermal Titration Calorimetry is presented.

  7. Antibody Affinity Maturation in Fishes—Our Current Understanding

    PubMed Central

    Magor, Brad G.

    2015-01-01

    It has long been believed that fish lack antibody affinity maturation, in part because they were thought to lack germinal centers. Recent research done on sharks and bony fishes indicates that these early vertebrates are able to affinity mature their antibodies. This article reviews the functionality of the fish homologue of the immunoglobulin (Ig) mutator enzyme activation-induced cytidine deaminase (AID). We also consider the protein and molecular evidence for Ig somatic hypermutation and antibody affinity maturation. In the context of recent evidence for a putative proto-germinal center in fishes we propose some possible reasons that observed affinity maturation in fishes often seems lacking and propose future work that might shed further light on this process in fishes. PMID:26264036

  8. Electrospun polyethersulfone affinity membrane: membrane preparation and performance evaluation.

    PubMed

    Ma, Zuwei; Lan, Zhengwei; Matsuura, Takeshi; Ramakrishna, Seeram

    2009-11-01

    Non-woven polyethersulfone (PES) membranes were prepared by electrospinning. After heat treatment and surface activation, the membranes were covalently functionalized with ligands to be used as affinity membranes. The membranes were characterized in terms of fiber diameter, porosity, specific area, pore size, ligand density and binding capacities. To evaluate the binding efficiency of the membrane, dynamic adsorption of bovine serum albumin (BSA) on the Cibacron blue F3GA (CB) functionalized PES membrane was studied. Experimental breakthrough curves were fitted with the theoretical curves based on the plate model to estimate plate height (H(p)) of the affinity membrane. The high value of H(p) (1.6-8 cm) of the affinity membrane implied a poor dynamic binding efficiency, which can be explained by the intrinsic microstructures of the material. Although the electrospun membrane might not be an ideal candidate for the preparative affinity membrane chromatography for large-scale production, it still can be used for fast small-scale protein purification in which a highly efficient binding is not required. Spin columns packed with protein A/G immobilized PES membranes were demonstrated to be capable of binding IgG specifically. SDS-PAGE results demonstrated that the PES affinity membrane had high specific binding selectivity for IgG molecules and low non-specific protein adsorption. Compared with other reported affinity membranes, the PES affinity membrane had a comparable IgG binding capacity of 4.5 mg/ml, and had a lower flow through pressure drop due to its larger pore size. In conclusion, the novel PES affinity membrane is an ideal spin column packing material for fast protein purification.

  9. Considering affinity: an ethereal conversation (part two of three).

    PubMed

    Winsor, Mary P

    2015-06-01

    In 1840 Hugh Strickland published a diagram showing the relationships of genera of birds in the kingfisher family. Three years later he applied this mapping idea to genera of birds of prey and songbirds, creating a large wall chart that he displayed to colleagues but never published. Both of his diagrams featured a scale of degrees of affinity. The meaning of taxonomic affinity was something Darwin thought about deeply. Details in the chart undermine Strickland's claim that his method was purely inductive.

  10. Proton affinity of methyl nitrate - Less than proton affinity of nitric acid

    NASA Technical Reports Server (NTRS)

    Lee, Timothy J.; Rice, Julia E.

    1992-01-01

    Several state-of-the-art ab initio quantum mechanical methods were used to investigate the equilibrium structure, dipole moments, harmonic vibrational frequencies, and IR intensities of methyl nitrate, methanol, and several structures of protonated methyl nitrate, using the same theoretical methods as in an earlier study (Lee and Rice, 1992) of nitric acid. The ab initio results for methyl nitrate and methanol were found to be in good agreement with available experimental data. The proton affinity (PA) of methyl nitrate was calculated to be 176.9 +/-5 kcal/mol, in excellent agreement with the experimental value 176 kcal/mol obtained by Attina et al. (1987) and less than the PA value of nitric acid. An explanation of the discrepancy of the present results with those of an earlier study on protonated nitric acid is proposed.

  11. Single event mass spectrometry

    DOEpatents

    Conzemius, Robert J.

    1990-01-16

    A means and method for single event time of flight mass spectrometry for analysis of specimen materials. The method of the invention includes pulsing an ion source imposing at least one pulsed ion onto the specimen to produce a corresponding emission of at least one electrically charged particle. The emitted particle is then dissociated into a charged ion component and an uncharged neutral component. The ion and neutral components are then detected. The time of flight of the components are recorded and can be used to analyze the predecessor of the components, and therefore the specimen material. When more than one ion particle is emitted from the specimen per single ion impact, the single event time of flight mass spectrometer described here furnis This invention was made with Government support under Contract No. W-7405-ENG82 awarded by the Department of Energy. The Government has certain rights in the invention.

  12. Sequential peptide affinity purification system for the systematic isolation and identification of protein complexes from Escherichia coli.

    PubMed

    Babu, Mohan; Butland, Gareth; Pogoutse, Oxana; Li, Joyce; Greenblatt, Jack F; Emili, Andrew

    2009-01-01

    Biochemical purification of affinity-tagged proteins in combination with mass spectrometry methods is increasingly seen as a cornerstone of systems biology, as it allows for the systematic genome-scale characterization of macromolecular protein complexes, representing demarcated sets of stably interacting protein partners. Accurate and sensitive identification of both the specific and shared polypeptide components of distinct complexes requires purification to near homogeneity. To this end, a sequential peptide affinity (SPA) purification system was developed to enable the rapid and efficient isolation of native Escherichia coli protein complexes (J Proteome Res 3:463-468, 2004). SPA purification makes use of a dual-affinity tag, consisting of three modified FLAG sequences (3X FLAG) and a calmodulin binding peptide (CBP), spaced by a cleavage site for tobacco etch virus (TEV) protease (J Proteome Res 3:463-468, 2004). Using the lambda-phage Red homologous recombination system (PNAS 97:5978-5983, 2000), a DNA cassette, encoding the SPA-tag and a selectable marker flanked by gene-specific targeting sequences, is introduced into a selected locus in the E. coli chromosome so as to create a C-terminal fusion with the protein of interest. This procedure aims for near-endogenous levels of tagged protein production in the recombinant bacteria to avoid spurious, non-specific protein associations (J Proteome Res 3:463-468, 2004). In this chapter, we describe a detailed, optimized protocol for the tagging, purification, and subsequent mass spectrometry-based identification of the subunits of even low-abundance bacterial protein complexes isolated as part of an ongoing large-scale proteomic study in E. coli (Nature 433:531-537, 2005).

  13. Direct measurement of equilibrium constants for high-affinity hemoglobins.

    PubMed

    Kundu, Suman; Premer, Scott A; Hoy, Julie A; Trent, James T; Hargrove, Mark S

    2003-06-01

    The biological functions of heme proteins are linked to their rate and affinity constants for ligand binding. Kinetic experiments are commonly used to measure equilibrium constants for traditional hemoglobins comprised of pentacoordinate ligand binding sites and simple bimolecular reaction schemes. However, kinetic methods do not always yield reliable equilibrium constants with more complex hemoglobins for which reaction mechanisms are not clearly understood. Furthermore, even where reaction mechanisms are clearly understood, it is very difficult to directly measure equilibrium constants for oxygen and carbon monoxide binding to high-affinity (K(D) < 1 micro M) hemoglobins. This work presents a method for direct measurement of equilibrium constants for high-affinity hemoglobins that utilizes a competition for ligands between the "target" protein and an array of "scavenger" hemoglobins with known affinities. This method is described for oxygen and carbon monoxide binding to two hexacoordinate hemoglobins: rice nonsymbiotic hemoglobin and Synechocystis hemoglobin. Our results demonstrate that although these proteins have different mechanisms for ligand binding, their affinities for oxygen and carbon monoxide are similar. Their large affinity constants for oxygen, 285 and approximately 100 micro M(-1) respectively, indicate that they are not capable of facilitating oxygen transport.

  14. Functionalized multi-walled carbon nanotubes as affinity ligands

    NASA Astrophysics Data System (ADS)

    Yu, L.; Li, C. M.; Zhou, Q.; Gan, Y.; Bao, Q. L.

    2007-03-01

    Functionalization of carbon nanotubes is very challenging for their applications. The paper here describes a new method to functionalize multi-walled carbon nanotubes (MWCNTs) as specific affinity adsorbents. MWCNTs were acid purified and pretreated with (3-aminopropyl)-triethoxysilane (APTES) in order to introduce abundant amino groups on the surface of MWCNTs. After the conversion of amino groups to carboxyl groups by succinic acid anhydride, MWCNTs were attached to protein A or aminodextran using 1-ethyl-3,3' (dimethylamion)-propylcarbodiimide as a biofunctional crosslinker. The incorporation of aminodextran as a spacer arm noticeably increased the binding capacity of the APTES-modified MWCNTs for protein A. The application of affinity MWCNTs for purification of immunoglobulin G was then evaluated. The affinity of MWCNTs with AMD spacer exhibited a high adsorption capacity of ~361 µg IgG/mg MWCNT (wet basis). About 75% of bound IgG was eluted from affinity MWCNTs (ANT-I and ANT-II) and ELISA confirmed that the biological activity of IgG was well preserved during the course of affinity separation. The functionalized MWCNTs could be potentially used in affinity chromatography.

  15. Local BLyS production by T follicular cells mediates retention of high affinity B cells during affinity maturation

    PubMed Central

    Goenka, Radhika; Matthews, Andrew H.; Zhang, Bochao; O’Neill, Patrick J.; Scholz, Jean L.; Migone, Thi-Sau; Leonard, Warren J.; Stohl, William; Hershberg, Uri

    2014-01-01

    We have assessed the role of B lymphocyte stimulator (BLyS) and its receptors in the germinal center (GC) reaction and affinity maturation. Despite ample BLyS retention on B cells in follicular (FO) regions, the GC microenvironment lacks substantial BLyS. This reflects IL-21–mediated down-regulation of the BLyS receptor TACI (transmembrane activator and calcium modulator and cyclophilin ligand interactor) on GC B cells, thus limiting their capacity for BLyS binding and retention. Within the GC, FO helper T cells (TFH cells) provide a local source of BLyS. Whereas T cell–derived BLyS is dispensable for normal GC cellularity and somatic hypermutation, it is required for the efficient selection of high affinity GC B cell clones. These findings suggest that during affinity maturation, high affinity clones rely on TFH-derived BLyS for their persistence. PMID:24367004

  16. Ion mobility-mass spectrometry.

    PubMed

    Kanu, Abu B; Dwivedi, Prabha; Tam, Maggie; Matz, Laura; Hill, Herbert H

    2008-01-01

    This review article compares and contrasts various types of ion mobility-mass spectrometers available today and describes their advantages for application to a wide range of analytes. Ion mobility spectrometry (IMS), when coupled with mass spectrometry, offers value-added data not possible from mass spectra alone. Separation of isomers, isobars, and conformers; reduction of chemical noise; and measurement of ion size are possible with the addition of ion mobility cells to mass spectrometers. In addition, structurally similar ions and ions of the same charge state can be separated into families of ions which appear along a unique mass-mobility correlation line. This review describes the four methods of ion mobility separation currently used with mass spectrometry. They are (1) drift-time ion mobility spectrometry (DTIMS), (2) aspiration ion mobility spectrometry (AIMS), (3) differential-mobility spectrometry (DMS) which is also called field-asymmetric waveform ion mobility spectrometry (FAIMS) and (4) traveling-wave ion mobility spectrometry (TWIMS). DTIMS provides the highest IMS resolving power and is the only IMS method which can directly measure collision cross-sections. AIMS is a low resolution mobility separation method but can monitor ions in a continuous manner. DMS and FAIMS offer continuous-ion monitoring capability as well as orthogonal ion mobility separation in which high-separation selectivity can be achieved. TWIMS is a novel method of IMS with a low resolving power but has good sensitivity and is well intergrated into a commercial mass spectrometer. One hundred and sixty references on ion mobility-mass spectrometry (IMMS) are provided.

  17. Mass spectrometry in environmental toxicology.

    PubMed

    Groh, Ksenia J; Suter, Marc J-F

    2014-01-01

    In environmental toxicology, mass spectrometry can be applied to evaluate both exposure to chemicals as well as their effects in organisms. Various ultra-trace techniques are employed today to measure pollutants in different environmental compartments. Increasingly, effect-directed analysis is being applied to focus chemical monitoring on sites of ecotoxicological concern. Mass spectrometry is also very instrumental for studying the interactions of chemicals with organisms on the molecular and cellular level, providing new insights into mechanisms of toxicity. In the future, diverse mass spectrometry-based techniques are expected to become even more widely used in this field, contributing to the refinement of currently used environmental risk assessment strategies.

  18. Nanoparticle multivalency counterbalances the ligand affinity loss upon PEGylation.

    PubMed

    Hennig, Robert; Pollinger, Klaus; Veser, Anika; Breunig, Miriam; Goepferich, Achim

    2014-11-28

    The conjugation of receptor ligands to shielded nanoparticles is a widely used strategy to precisely control nanoparticle-cell interactions. However, it is often overlooked that a ligand's affinity can be severely impaired by its attachment to the polyethylene glycol (PEG) chains that are frequently used to protect colloids from serum protein adsorption. Using the model ligand EXP3174, a small-molecule antagonist for the angiotensin II receptor type 1 (AT1R), we investigated the ligand's affinity before and after its PEGylation and when attached to PEGylated nanoparticles. The PEGylated ligand displayed a 580-fold decreased receptor affinity compared to the native ligand. Due to their multivalency, the nanoparticles regained a low nanomolar receptor affinity, which is in the range of the affinity of the native ligand. Moreover, a four orders of magnitude higher concentration of free ligand was required to displace PEGylated nanoparticles carrying EXP3174 from the receptor. On average, one nanoparticle was decorated with 11.2 ligand molecules, which led to a multivalent enhancement factor of 22.5 compared to the monovalent PEGylated ligand. The targeted nanoparticles specifically bound the AT1R and showed no interaction to receptor negative cells. Our study shows that the attachment of a small-molecule ligand to a PEG chain can severely affect its receptor affinity. Concomitantly, when the ligand is tethered to nanoparticles, the immense avidity greatly increases the ligand-receptor interaction. Based on our results, we highly recommend the affinity testing of receptor ligands before and after PEGylation to identify potent molecules for active nanoparticle targeting.

  19. Gradient-based habitat affinities predict species vulnerability to drought.

    PubMed

    Debinski, Diane M; Caruthers, Jennet C; Cook, Dianne; Crowley, Jason; Wickham, Hadley

    2013-05-01

    Ecological fingerprints of climate change are becoming increasingly evident at broad geographical scales as measured by species range shifts and changes in phenology. However, finer-scale species-level responses to environmental fluctuations may also provide an important bellwether of impending future community responses. Here we examined changes in abundance of butterfly species along a hydrological gradient of six montane meadow habitat types in response to drought. Our data collection began prior to the drought, and we were able to track changes for 11 years, of which eight were considered mild to extreme drought conditions. We separated the species into those that had an affinity for hydric vs. xeric habitats. We suspected that drought would favor species with xeric habitat affinities, but that there could be variations in species-level responses along the hydrological gradient. We also suspected that mesic meadows would be most sensitive to drought conditions. Temporal trajectories were modeled for both species groups (hydric vs. xeric affinity) and individual species. Abundances of species with affinity for xeric habitats increased in virtually all meadow types. Conversely, abundances of species with affinity for hydric habitats decreased, particularly in mesic and xeric meadows. Mesic meadows showed the most striking temporal abundance trajectory: Increasing abundances of species with xeric habitat affinity were offset by decreasing or stable abundances of species with hydric habitat affinity. The one counterintuitive finding was that, in some hydric meadows, species with affinity for hydric habitats increased. In these cases, we suspect that decreasing moisture conditions in hydric meadows actually increased habitat suitability because sites near the limit of moisture extremes for some species became more acceptable. Thus, species responses were relatively predictable based upon habitat affinity and habitat location along the hydrological gradient, and

  20. Methods for Neutron Spectrometry

    DOE R&D Accomplishments Database

    Brockhouse, Bertram N.

    1961-01-09

    The appropriate theories and the general philosophy of methods of measurement and treatment of data neutron spectrometry are discussed. Methods of analysis of results for liquids using the Van Hove formulation, and for crystals using the Born-von Karman theory, are reviewed. The most useful of the available methods of measurement are considered to be the crystal spectrometer methods and the pulsed monoenergetic beam/time-of-flight method. Pulsed-beam spectrometers have the advantage of higher counting rates than crystal spectrometers, especially in view of the fact that simultaneous measurements in several counters at different angles of scattering are possible in pulsed-beam spectrometers. The crystal spectrometer permits several valuable new types of specialized experiments to be performed, especially energy distribution measurements at constant momentum transfer. The Chalk River triple-axis crystal-spectrometer is discussed, with reference to its use in making the specialized experiments. The Chalk River rotating crystal (pulsed-beam) spectrometer is described, and a comparison of this type instrument with other pulsed-beam spectrometers is made. A partial outline of the theory of operation of rotating-crystal spectrometers is presented. The use of quartz-crystal filters for fast neutron elimination and for order elimination is discussed. (auth)

  1. Gridless Overtone Mobility Spectrometry

    PubMed Central

    Zucker, Steven M.; Ewing, Michael A.; Clemmer, David E.

    2013-01-01

    A novel overtone mobility spectrometry (OMS) instrument utilizing a gridless elimination mechanism and cooperative radio frequency confinement is described. The gridless elimination region uses a set of mobility-discriminating radial electric fields that are designed so that the frequency of field application results in selective transmission and elimination of ions. To neutralize ions with mobilities that do not match the field application frequency, active elimination regions radially defocus ions towards the lens walls. Concomitantly, a lens-dependent radio frequency waveform is applied to the transmission regions of the drift tube resulting in radial confinement for mobility-matched ions. Compared with prior techniques, which use many grids for ion elimination, the new gridless configuration substantially reduces indiscriminate ion losses. A description of the apparatus and elimination process, including detailed simulations showing how ions are transmitted and eliminated is presented. A prototype 28 cm long OMS instrument is shown to have a resolving power of 20 and is capable of attomole detection limits of a model peptide (angiotensin I) spiked into a complex mixture (in this case peptides generated from digestion of β-casein with trypsin). PMID:24125033

  2. Accelerator mass spectrometry.

    PubMed

    Hellborg, Ragnar; Skog, Göran

    2008-01-01

    In this overview the technique of accelerator mass spectrometry (AMS) and its use are described. AMS is a highly sensitive method of counting atoms. It is used to detect very low concentrations of natural isotopic abundances (typically in the range between 10(-12) and 10(-16)) of both radionuclides and stable nuclides. The main advantages of AMS compared to conventional radiometric methods are the use of smaller samples (mg and even sub-mg size) and shorter measuring times (less than 1 hr). The equipment used for AMS is almost exclusively based on the electrostatic tandem accelerator, although some of the newest systems are based on a slightly different principle. Dedicated accelerators as well as older "nuclear physics machines" can be found in the 80 or so AMS laboratories in existence today. The most widely used isotope studied with AMS is 14C. Besides radiocarbon dating this isotope is used in climate studies, biomedicine applications and many other fields. More than 100,000 14C samples are measured per year. Other isotopes studied include 10Be, 26Al, 36Cl, 41Ca, 59Ni, 129I, U, and Pu. Although these measurements are important, the number of samples of these other isotopes measured each year is estimated to be less than 10% of the number of 14C samples.

  3. Biomedical accelerator mass spectrometry

    NASA Astrophysics Data System (ADS)

    Freeman, Stewart P. H. T.; Vogel, John S.

    1995-05-01

    Ultrasensitive SIMS with accelerator based spectrometers has recently begun to be applied to biomedical problems. Certain very long-lived radioisotopes of very low natural abundances can be used to trace metabolism at environmental dose levels ( [greater-or-equal, slanted] z mol in mg samples). 14C in particular can be employed to label a myriad of compounds. Competing technologies typically require super environmental doses that can perturb the system under investigation, followed by uncertain extrapolation to the low dose regime. 41Ca and 26Al are also used as elemental tracers. Given the sensitivity of the accelerator method, care must be taken to avoid contamination of the mass spectrometer and the apparatus employed in prior sample handling including chemical separation. This infant field comprises the efforts of a dozen accelerator laboratories. The Center for Accelerator Mass Spectrometry has been particularly active. In addition to collaborating with groups further afield, we are researching the kinematics and binding of genotoxins in-house, and we support innovative uses of our capability in the disciplines of chemistry, pharmacology, nutrition and physiology within the University of California. The field can be expected to grow further given the numerous potential applications and the efforts of several groups and companies to integrate more the accelerator technology into biomedical research programs; the development of miniaturized accelerator systems and ion sources capable of interfacing to conventional HPLC and GMC, etc. apparatus for complementary chemical analysis is anticipated for biomedical laboratories.

  4. Coenzyme-like ligands for affinity isolation of cholesterol oxidase.

    PubMed

    Xin, Yu; Lu, Liushen; Wang, Qing; Zhang, Ling; Tong, Yanjun; Wang, Wu

    2016-05-15

    Two coenzyme-like chemical ligands were designed and synthesized for affinity isolation of cholesterol oxidase (COD). To simulate the structure of natural coenzyme of COD (flavin adenine dinucleotide (FAD)), on Sepharose beads, 5-aminouracil, cyanuric chloride and 1, 4-butanediamine were composed and then modified. The COD gene from Brevibacterium sp. (DQ345780) was expressed in Escherichia coli BL21 (DE3), and then the sorbents were applied to adsorption analysis with the pure enzyme. Subsequently, the captured enzyme was applied to SDS-PAGE and activity analysis. As calculated, the theoretical maximum adsorption (Qmax) of the two affinity sorbents (RL-1 and RL-2) were ∼83.5 and 46.3mg/g wet gel; and the desorption constant Kd of the two sorbents were ∼6.02×10(-4) and 1.19×10(-4)μM. The proteins after cell lysis were applied to affinity isolation, and then after one step of affinity binding on the two sorbents, the protein recoveries of RL-1 and RL-2 were 9.2% and 9.7%; the bioactivity recoveries were 92.7% and 91.3%, respectively. SDS-PAGE analysis revealed that the purities of COD isolated with the two affinity sorbents were approximately 95%.

  5. Improving antibody binding affinity and specificity for therapeutic development.

    PubMed

    Bostrom, Jenny; Lee, Chingwei V; Haber, Lauric; Fuh, Germaine

    2009-01-01

    Affinity maturation is an important part of the therapeutic antibody development process as in vivo activity often requires high binding affinity. Here, we describe a targeted approach for affinity improvement of therapeutic antibodies. Sets of CDR residues that are solvent accessible and relatively diverse in natural antibodies are targeted for diversification. Degenerate oligonucleotides are used to generate combinatorial phage-displayed antibody libraries with varying degree of diversity at randomized positions from which high-affinity antibodies can be selected. An advantage of using antibodies for therapy is their exquisite target specificity, which enables selective antigen binding and reduces off-target effects. However, it can be useful, and often it is necessary, to generate cross-reactive antibodies binding to not only the human antigen but also the corresponding non-human primate or rodent orthologs. Such cross-reactive antibodies can be used to validate the therapeutic targeting and examine the safety profile in preclinical animal models before committing to a costly development track. We show how affinity improvement and cross-species binding can be achieved in a one-step process.

  6. High-affinity K+ uptake in pepper plants.

    PubMed

    Martínez-Cordero, M Angeles; Martínez, Vicente; Rubio, Francisco

    2005-06-01

    High-affinity K+ uptake is an essential process for plant nutrition under K+-limiting conditions. The results presented here demonstrate that pepper (Capsicum annuum) plants grown in the absence of NH4+ and starved of K+ show an NH4+-sensitive high-affinity K+ uptake that allows plant roots to deplete external K+ to values below 1 microM. When plants are grown in the presence of NH4+, high-affinity K+ uptake is not inhibited by NH4+. Although NH4+-grown plants deplete external K+ below 1 microM in the absence of NH4+, when 1 mM NH4+ is present they do not deplete external K+ below 10 microM. A K+ transporter of the HAK family, CaHAK1, is very likely mediating the NH4+-sensitive component of the high-affinity K+ uptake in pepper roots. CaHAK1 is strongly induced in the roots that show the NH4+-sensitive high-affinity K+ uptake and its induction is reduced in K+-starved plants grown in the presence of NH4+. The NH4+-insensitive K+ uptake may be mediated by an AKT1-like K+ channel.

  7. Affine kinematics in planar fibrous connective tissues: an experimental investigation.

    PubMed

    Jayyosi, C; Affagard, J-S; Ducourthial, G; Bonod-Bidaud, C; Lynch, B; Bancelin, S; Ruggiero, F; Schanne-Klein, M-C; Allain, J-M; Bruyère-Garnier, K; Coret, M

    2017-03-29

    The affine transformation hypothesis is usually adopted in order to link the tissue scale with the fibers scale in structural constitutive models of fibrous tissues. Thanks to the recent advances in imaging techniques, such as multiphoton microscopy, the microstructural behavior and kinematics of fibrous tissues can now be monitored at different stretching within the same sample. Therefore, the validity of the affine hypothesis can be investigated. In this paper, the fiber reorientation predicted by the affine assumption is compared to experimental data obtained during mechanical tests on skin and liver capsule coupled with microstructural imaging using multiphoton microscopy. The values of local strains and the collagen fibers orientation measured at increasing loading levels are used to compute a theoretical estimation of the affine reorientation of collagen fibers. The experimentally measured reorientation of collagen fibers during loading could not be successfully reproduced with this simple affine model. It suggests that other phenomena occur in the stretching process of planar fibrous connective tissues, which should be included in structural constitutive modeling approaches.

  8. Biphasic Affinity Chromatographic Approach for Deep Tyrosine Phosphoproteome Analysis.

    PubMed

    Deng, Zhenzhen; Dong, Mingming; Wang, Yan; Dong, Jing; Li, Shawn S-C; Zou, Hanfa; Ye, Mingliang

    2017-02-21

    Tyrosine phosphorylation (pTyr) is important for normal physiology and implicated in many human diseases, particularly cancer. Identification of pTyr sites is critical to dissecting signaling pathways and understanding disease pathologies. However, compared with serine/threonine phosphorylation (pSer/pThr), the analysis of pTyr at the proteome level is more challenging due to its low abundance. Here, we developed a biphasic affinity chromatographic approach where Src SH2 superbinder was coupled with NeutrAvidin affinity chromatography, for tyrosine phosphoproteome analysis. With the use of competitive elution agent biotin-pYEEI, this strategy can distinguish high-affinity phosphotyrosyl peptides from low-affinity ones, while the excess competitive agent is readily removed by using NeutrAvidin agarose resin in an integrated tip system. The excellent performance of this system was demonstrated by analyzing tyrosine phosphoproteome of Jurkat cells from which 3,480 unique pTyr sites were identified. The biphasic affinity chromatography method for deep Tyr phosphoproteome analysis is rapid, sensitive, robust, and cost-effective. It is widely applicable to the global analysis of the tyrosine phosphoproteome associated with tyrosine kinase signal transduction.

  9. Protein purification by aminosquarylium cyanine dye-affinity chromatography.

    PubMed

    Silva, M S; Graça, V C; Reis, L V; Santos, P F; Almeida, P; Queiroz, J A; Sousa, F

    2013-12-01

    The most selective purification method for proteins and other biomolecules is affinity chromatography. This method is based on the unique biological-based specificity of the biomolecule-ligand interaction and commonly uses biological ligands. However, these ligands may present some drawbacks, mainly because of their cost and lability. Dye-affinity chromatography overcomes the limitations of biological ligands and is widely used owing to the low cost of synthetic dyes and to their resistance to biological and chemical degradation. In this work, immobilized aminosquarylium cyanine dyes are used in order to exploit affinity interactions with standard proteins such as lysozyme, α-chymotrypsin and trypsin. These studies evaluate the affinity interactions occurring between the immobilized ligand and the different proteins, as a reflection of the sum of several molecular interactions, namely ionic, hydrophobic and van der Waals, spread throughout the structure, in a defined spatial manner. The results show the possibility of using an aminosquarylium cyanine dye bearing a N-hexyl pendant chain, with a ligand density of 1.8 × 10(-2) mmol of dye/g of chromatographic support, to isolate lysozyme, α-chymotrypsin and trypsin from a mixture. The application of a decreasing ammonium sulfate gradient resulted in the recovery of lysozyme in the flowthrough. On the other hand, α-chymotrypsin and trypsin were retained, involving different interactions with the ligand. In conclusion, this study demonstrates the potential applicability of ligands such as aminosquarylium cyanine dyes for the separation and purification of proteins by affinity chromatography.

  10. Development of Substrate-Selective Probes for Affinity Pulldown of Histone Demethylases

    PubMed Central

    2015-01-01

    JmjC-domain containing histone demethylases (JHDMs) play critical roles in many key cellular processes and have been implicated in multiple disease conditions. Each enzyme within this family is known to have a strict substrate scope, specifically the position of the lysine within the histone and its degree of methylation. While much progress has been made in determining the substrates of each enzyme, new methods with which to systematically profile each histone mark are greatly needed. Novel chemical tools have the potential to fill this role and, furthermore, can be used as probes to answer fundamental questions about these enzymes and serve as potential therapeutic leads. In this work, we first investigated three small-molecule probes differing in the degree of “methylation state” and their differential bindings to JHDM1A (an H3K36me1/2 demethylase) using a fluorescence polarization-based competition assay. We then applied this specificity toward the “methylation state” and combined it with specificity toward lysine position in the design and synthesis of a peptidic probe targeting H3K36me2 JHDMs. The probe is further functionalized with a benzophenone cross-linking moiety and a biotin for affinity purification. Results showed binding of the peptidic probe to JHDM1A and specific enrichment of this protein in the presence of its native histone substrates. Affinity purification pulldown experiments from nuclear lysate coupled with mass spectrometry revealed the capability of the probe to pull out and enrich JHDMs along with other epigenetic proteins and transcriptional regulators. PMID:25335116

  11. Preparation of multiprotein complexes from Arabidopsis chloroplasts using tandem affinity purification.

    PubMed

    Andrès, Charles; Agne, Birgit; Kessler, Felix

    2011-01-01

    Since its first description in 1998 (Rigaut et al., Nat Biotech 17:1030-1032, 1999), the TAP method, for Tandem Affinity Purification, has become one of the most popular methods for the purification of in vivo protein complexes and the identification of their composition by subsequent mass spectrometry analysis. The TAP method is based on the use of a tripartite tag fused to a target protein expressed in the organism of interest. A TAP tag has two independent binding regions separated by a protease cleavage site, and therefore allows two successive affinity purification steps. The most common TAP tag consists of two IgG binding repeats of Protein A from Staphylococcus aureus (ProtA) separated from a calmodulin-binding peptide by a Tobacco Etch Virus (TEV) protease cleavage site. Using the TAP method, native protein complexes can be purified efficiently with a reduced contaminant background when compared to single step purification methods. Initially developed in the yeast model system, the TAP method has been adapted to most common model organisms. The first report of the purification of protein complexes from plant tissue by the TAP method was published in 2004 by Rohila et al. (Plant J 38:172-181, 2004). The synthetic TAP tag gene described in this study has been optimized for use in plants, and since then, has been successfully used from single gene analyses to high-throughput studies of whole protein families (Rohila et al., PLoS ONE 4:e6685, 2009). Here, we describe a TAP tag purification method for the purification of protein complexes from total Arabidopsis extracts, that we employed successfully using a TAP-tagged chloroplast outer envelope protein.

  12. The affine structure of gravitational theories: Symplectic groups and geometry

    NASA Astrophysics Data System (ADS)

    Capozziello, Salvatore; Cirilo-Lombardo, D. J.; de Laurentis, Mariafelicia

    2014-09-01

    We give a geometrical description of gravitational theories from the viewpoint of symmetries and affine structure. We show how gravity, considered as a gauge theory, can be consistently achieved by the nonlinear realization of the conformal-affine group in an indirect manner: due to the partial isomorphism between CA(3, 1) and the centrally extended Sp( 8), we perform a nonlinear realization of the centrally extended (CE)Sp( 8) in its semi-simple version. In particular, starting from the bundle structure of gravity, we derive the conformal-affine Lie algebra and then, by the nonlinear realization, we define the coset field transformations, the Cartan forms and the inverse Higgs constraints. Finally, we discuss the geometrical Lagrangians where all the information on matter fields and their interactions can be contained.

  13. Affinity maturation of antibodies requires integrity of the adult thymus.

    PubMed

    AbuAttieh, Mouhammed; Bender, Diane; Liu, Esther; Wettstein, Peter; Platt, Jeffrey L; Cascalho, Marilia

    2012-02-01

    The generation of B-cell responses to proteins requires a functional thymus to produce CD4(+) T cells which helps in the activation and differentiation of B cells. Because the mature T-cell repertoire has abundant cells with the helper phenotype, one might predict that in mature individuals, the generation of B-cell memory would proceed independently of the thymus. Contrary to that prediction, we show here that the removal of the thymus after the establishment of the T-cell compartment or sham surgery without removal of the thymus impairs the affinity maturation of antibodies. Because removal or manipulation of the thymus did not decrease the frequency of mutation of the Ig variable heavy chain exons encoding antigen-specific antibodies, we conclude that the thymus controls affinity maturation of antibodies in the mature individual by facilitating the selection of B cells with high-affinity antibodies.

  14. A fast quantum algorithm for the affine Boolean function identification

    NASA Astrophysics Data System (ADS)

    Younes, Ahmed

    2015-02-01

    Bernstein-Vazirani algorithm (the one-query algorithm) can identify a completely specified linear Boolean function using a single query to the oracle with certainty. The first aim of the paper is to show that if the provided Boolean function is affine, then one more query to the oracle (the two-query algorithm) is required to identify the affinity of the function with certainty. The second aim of the paper is to show that if the provided Boolean function is incompletely defined, then the one-query and the two-query algorithms can be used as bounded-error quantum polynomial algorithms to identify certain classes of incompletely defined linear and affine Boolean functions respectively with probability of success at least 2/3.

  15. Enhancing Community Detection By Affinity-based Edge Weighting Scheme

    SciTech Connect

    Yoo, Andy; Sanders, Geoffrey; Henson, Van; Vassilevski, Panayot

    2015-10-05

    Community detection refers to an important graph analytics problem of finding a set of densely-connected subgraphs in a graph and has gained a great deal of interest recently. The performance of current community detection algorithms is limited by an inherent constraint of unweighted graphs that offer very little information on their internal community structures. In this paper, we propose a new scheme to address this issue that weights the edges in a given graph based on recently proposed vertex affinity. The vertex affinity quantifies the proximity between two vertices in terms of their clustering strength, and therefore, it is ideal for graph analytics applications such as community detection. We also demonstrate that the affinity-based edge weighting scheme can improve the performance of community detection algorithms significantly.

  16. On the electron affinity of the oxygen atom

    NASA Technical Reports Server (NTRS)

    Bauschlicher, C. W., Jr.; Langhoff, S. R.; Partridge, H.; Taylor, P. R.

    1986-01-01

    The electron affinity (EA) of oxygen is computed to be 1.287 eV, using 2p electron full configuration-interaction (CI) wave functions expanded in a 6s5p3d2f Slater-type orbital basis. The best complete active space self-consistent field - multireference CI (CASSCF-MRCI) result including only 2p correlation is 1.263 eV. However, inclusion of 2s intrashell and 2s2p intershell correlation increases the computed EA to 1.290 at the CASSCF-MRCI level. At the full CI basis set limit, the 2s contribution to the electron affinity is estimated to be as large as 0.1 eV. This study clearly establishes the synergistic effect between the higher excitations and basis set completeness on the electron affinity when the 2s electrons are correlated.

  17. On the electron affinity of the oxygen atom

    NASA Technical Reports Server (NTRS)

    Bauschlicher, C. W., Jr.; Langhoff, S. R.; Partridge, H.; Taylor, P. R.

    1986-01-01

    The electron affinity of oxygen is computed to be 1.287 eV, at the full CI level using a 6s5p3d 2f Slater-type orbital basis and correlating only the 2p electrons. The best CASSCF-MRCI result including only 2p correlation is 1.263 eV. However, inclusion of 2s intrashell and 2s2p intershell coorelation increases the computed EA to 1.290 eV at the CASSCF-MRCI level. At the full CI basis set limit, the 2s contribution to the electron affinity is estimated to be as large as 0.1 eV. The higher excitation contribution to the electron affinity is found to increase substantially with basis set completeness, especially when the 2s electrons are correlated. Relativistic effects are shown to make a small (less than 0.01 eV) change in the EA.

  18. AMPK beta subunits display isoform specific affinities for carbohydrates.

    PubMed

    Koay, Ann; Woodcroft, Ben; Petrie, Emma J; Yue, Helen; Emanuelle, Shane; Bieri, Michael; Bailey, Michael F; Hargreaves, Mark; Park, Jong-Tae; Park, Kwan-Hwa; Ralph, Stuart; Neumann, Dietbert; Stapleton, David; Gooley, Paul R

    2010-08-04

    AMP-activated protein kinase (AMPK) is a heterotrimer of catalytic (alpha) and regulatory (beta and gamma) subunits with at least two isoforms for each subunit. AMPK beta1 is widely expressed whilst AMPK beta2 is highly expressed in muscle and both beta isoforms contain a mid-molecule carbohydrate-binding module (beta-CBM). Here we show that beta2-CBM has evolved to contain a Thr insertion and increased affinity for glycogen mimetics with a preference for oligosaccharides containing a single alpha-1,6 branched residue. Deletion of Thr-101 reduces affinity for single alpha-1,6 branched oligosaccharides by 3-fold, while insertion of this residue into the equivalent position in the beta1-CBM sequence increases affinity by 3-fold, confirming the functional importance of this residue.

  19. Affine generalization of the Komar complex of general relativity

    NASA Astrophysics Data System (ADS)

    Mielke, Eckehard W.

    2001-02-01

    On the basis of the ``on shell'' Noether identities of the metric-affine gauge approach of gravity, an affine superpotential is derived which comprises the energy- and angular-momentum content of exact solutions. In the special case of general relativity (GR) or its teleparallel equivalent, the Komar or Freud complex, respectively, are recovered. Applying this to the spontaneously broken anti-de Sitter gauge model of McDowell and Mansouri with an induced Euler term automatically yields the correct mass and spin of the Kerr-AdS solution of GR with a (induced) cosmological constant without the factor two discrepancy of the Komar formula.

  20. A quantum affine algebra for the deformed Hubbard chain

    NASA Astrophysics Data System (ADS)

    Beisert, Niklas; Galleas, Wellington; Matsumoto, Takuya

    2012-09-01

    The integrable structure of the one-dimensional Hubbard model is based on Shastry's R-matrix and the Yangian of a centrally extended \\mathfrak {sl}(2|2) superalgebra. Alcaraz and Bariev have shown that the model admits an integrable deformation whose R-matrix has recently been found. This R-matrix is of trigonometric type and here we derive its underlying exceptional quantum affine algebra. We also show how the algebra reduces to the above-mentioned Yangian and to the conventional quantum affine \\mathfrak {sl}(2|2) algebra in two special limits.

  1. Isolation and mass spectrometry of transcription factor complexes.

    PubMed

    Sebastiaan Winkler, G; Lacomis, Lynne; Philip, John; Erdjument-Bromage, Hediye; Svejstrup, Jesper Q; Tempst, Paul

    2002-03-01

    Protocols are described that enable the isolation of novel proteins associated with a known protein and the subsequent identification of these proteins by mass spectrometry. We review the basics of nanosample handling and of two complementary approaches to mass analysis, and provide protocols for the entire process. The protein isolation procedure is rapid and based on two high-affinity chromatography steps. The method does not require previous knowledge of complex composition or activity and permits subsequent biochemical characterization of the isolated factor. As an example, we provide the procedures used to isolate and analyze yeast Elongator, a histone acetyltransferase complex important for transcript elongation, which led to the identification of three novel subunits.

  2. Student Engagement and Neoliberalism: Mapping an Elective Affinity

    ERIC Educational Resources Information Center

    Zepke, Nick

    2015-01-01

    The purpose of this article is to argue that student engagement, an important area for research about learning and teaching in formal higher education, has an elective affinity with neoliberalism, a hegemonic ideology in many countries of the developed world. The paper first surveys an extensive research literature examining student engagement and…

  3. "The Hunger Games": Literature, Literacy, and Online Affinity Spaces

    ERIC Educational Resources Information Center

    Curwood, Jen Scott

    2013-01-01

    This article examines adolescent literacy practices related to "The Hunger Games," a young adult novel and the first of a trilogy. By focusing on the interaction of social identities, discourses, and media paratexts within an online affinity space, this ethnographic study offers insight into how young adults engage with contemporary…

  4. Affinities and beyond! Developing Ways of Seeing in Online Spaces

    ERIC Educational Resources Information Center

    Davies, Julia

    2006-01-01

    This article presents an insider view of an online community of adults involved in sharing digital photography through a host website, Flickr. It describes how reciprocal teaching and learning partnerships in a dynamic multimodal environment are achieved through the creation of a "Third Space" or "Affinity Space", where "Funds of Knowledge" are…

  5. Electrochemical affinity biosensors for detection of mycotoxins: A review.

    PubMed

    Vidal, Juan C; Bonel, Laura; Ezquerra, Alba; Hernández, Susana; Bertolín, Juan R; Cubel, Carlota; Castillo, Juan R

    2013-11-15

    This review discusses the current state of electrochemical biosensors in the determination of mycotoxins in foods. Mycotoxins are highly toxic secondary metabolites produced by molds. The acute toxicity of these results in serious human and animal health problems, although it has been only since early 1960s when the first studied aflatoxins were found to be carcinogenic. Mycotoxins affect a broad range of agricultural products, most important cereals and cereal-based foods. A majority of countries, mentioning especially the European Union, have established preventive programs to control contamination and strict laws of the permitted levels in foods. Official methods of analysis of mycotoxins normally requires sophisticated instrumentation, e.g. liquid chromatography with fluorescence or mass detectors, combined with extraction procedures for sample preparation. For about sixteen years, the use of simpler and faster analytical procedures based on affinity biosensors has emerged in scientific literature as a very promising alternative, particularly electrochemical (i.e., amperometric, impedance, potentiometric or conductimetric) affinity biosensors due to their simplicity and sensitivity. Typically, electrochemical biosensors for mycotoxins use specific antibodies or aptamers as affinity ligands, although recombinant antibodies, artificial receptors and molecular imprinted polymers show potential utility. This article deals with recent advances in electrochemical affinity biosensors for mycotoxins and covers complete literature from the first reports about sixteen years ago.

  6. Toward an Affinity Space Methodology: Considerations for Literacy Research

    ERIC Educational Resources Information Center

    Lammers, Jayne C.; Curwood, Jen Scott; Magnifico, Alecia Marie

    2012-01-01

    As researchers seek to make sense of young people's online literacy practices and participation, questions of methodology are important to consider. In our work to understand the culture of physical, virtual and blended spheres that adolescents inhabit, we find it necessary to expand Gee's (2004) notion of affinity spaces. In this article, we draw…

  7. A molecular determinant of phosphoinositide affinity in mammalian TRPV channels

    PubMed Central

    Velisetty, Phanindra; Borbiro, Istvan; Kasimova, Marina A.; Liu, Luyu; Badheka, Doreen; Carnevale, Vincenzo; Rohacs, Tibor

    2016-01-01

    Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is an important cofactor for ion channels. Affinity for this lipid is a major determinant of channel inhibition by depletion of PI(4,5)P2 upon phospholipase C (PLC) activation. Little is known about what determines PI(4,5)P2 affinity in mammalian ion channels. Here we report that two members of the Transient Receptor Potential Vanilloid (TRPV) ion channel family, TRPV5 and TRPV6 lack a positively charged residue in the TM4-TM5 loop that was shown to interact with PI(4,5)P2 in TRPV1, which shows high affinity for this lipid. When this positively charged residue was introduced to either TRPV6 or TRPV5, they displayed markedly higher affinities for PI(4,5)P2, and were largely resistant to inhibition by PI(4,5)P2 depletion. Furthermore, Ca2+-induced inactivation of TRPV6 was essentially eliminated in the G488R mutant, showing the importance of PLC-mediated PI(4,5)P2 depletion in this process. Computational modeling shows that the introduced positive charge interacts with PI(4,5)P2 in TRPV6. PMID:27291418

  8. Kinetic Studies of Biological Interactions By Affinity Chromatography

    PubMed Central

    Schiel, John E.; Hage, David S.

    2009-01-01

    The rates at which biological interactions occur can provide important information on the mechanism and behavior of such processes in living systems. This review will discuss how affinity chromatography can be used as a tool to examine the kinetics of biological interactions. This approach, referred to here as biointeraction chromatography, uses a column with an immobilized binding agent to examine the association or dissociation of this agent with other compounds. The use of HPLC-based affinity columns in kinetic studies has received particular attention in recent years. Advantages of using HPLC with affinity chromatography for this purpose include the ability to reuse the same ligand within a column for a large number of experiments, and the good precision and accuracy of this approach. A number of techniques are available for kinetic studies through the use of affinity columns and biointeraction chromatography. These approaches include plate height measurements, peak profiling, peak fitting, split-peak measurements, and peak decay analysis. The general principles for each of these methods are discussed in this review and some recent applications of these techniques are presented. The advantages and potential limitations of each approach are also considered. PMID:19391173

  9. Affinity of cefoperazone for penicillin-binding proteins.

    PubMed Central

    Matsubara, N; Minami, S; Matsuhashi, M; Takaoka, M; Mitsuhashi, S

    1980-01-01

    Cefoperazone (T-1551, CFP) a new semisynthetic cephalosporin, has a broad spectrum of antibacterial activity. We investigated the affinity of CFP to penicillin-binding proteins (PBPs) and the inhibition of peptidoglycan synthesis by CFP. CFP had high affinities for Escherichia coli PBP-3, -1Bs, -2, and -1A, in descending order, and low affinities for PBP-4, -5, and -6. Similarly, CFP showed high affinity for Pseudomonas aeruginosa PBP-3, -1A, -1B, -2, and -4, in descending order. It is known that E. coli PBP-3 and P. aeruginosa PBP-3 participate in cell division. These results are in good agreement with the formation of filamentous cells of E. coli and P. aeruginosa treated with CFP. CFP had lower inhibitory activities on D-alanine carboxypeptidase IA and IB of E. coli than that of penicillin G, but its inhibitory activities on the cross-link formation in peptidoglycan synthesis were the same as those of penicillin G and higher than those of ampicillin. Images PMID:6448021

  10. A molecular determinant of phosphoinositide affinity in mammalian TRPV channels

    NASA Astrophysics Data System (ADS)

    Velisetty, Phanindra; Borbiro, Istvan; Kasimova, Marina A.; Liu, Luyu; Badheka, Doreen; Carnevale, Vincenzo; Rohacs, Tibor

    2016-06-01

    Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is an important cofactor for ion channels. Affinity for this lipid is a major determinant of channel inhibition by depletion of PI(4,5)P2 upon phospholipase C (PLC) activation. Little is known about what determines PI(4,5)P2 affinity in mammalian ion channels. Here we report that two members of the Transient Receptor Potential Vanilloid (TRPV) ion channel family, TRPV5 and TRPV6 lack a positively charged residue in the TM4-TM5 loop that was shown to interact with PI(4,5)P2 in TRPV1, which shows high affinity for this lipid. When this positively charged residue was introduced to either TRPV6 or TRPV5, they displayed markedly higher affinities for PI(4,5)P2, and were largely resistant to inhibition by PI(4,5)P2 depletion. Furthermore, Ca2+-induced inactivation of TRPV6 was essentially eliminated in the G488R mutant, showing the importance of PLC-mediated PI(4,5)P2 depletion in this process. Computational modeling shows that the introduced positive charge interacts with PI(4,5)P2 in TRPV6.

  11. Limit measures for affine cellular automata on topological Markov subgroups

    NASA Astrophysics Data System (ADS)

    Maass, Alejandro; Martínez, Servet; Sobottka, Marcelo

    2006-09-01

    Consider a topological Markov subgroup which is ps-torsion (with p prime) and an affine cellular automaton defined on it. We show that the Cesàro mean of the iterates, by the automaton of a probability measure with complete connections and summable memory decay that is compatible with the topological Markov subgroup, converges to the Haar measure.

  12. Affinity through Mathematical Activity: Cultivating Democratic Learning Communities

    ERIC Educational Resources Information Center

    Sengupta-Irving, Tesha

    2014-01-01

    In this article, the author demonstrates how a broader view of what shapes affinity is ideologically and practically linked to creating democratic learning communities. Specifically, the author explores how a teacher employed complex instruction (an equity pedagogy) with her ethnically and racially diverse students in the "lowest track"…

  13. Novel trends in affinity biosensors: current challenges and perspectives

    NASA Astrophysics Data System (ADS)

    Arugula, Mary A.; Simonian, Aleksandr

    2014-03-01

    Molecular biorecognition processes facilitate physical and biochemical interactions between molecules in all crucial metabolic pathways. Perhaps the target analyte and the biorecognition element interactions have the most impactful use in biosensing applications. Traditional analytical sensing systems offer excellent biorecognition elements with the ability to detect and determine the presence of analytes. High affinity antibodies and DNA play an important role in the development of affinity biosensors based on electrochemical, optical and mass sensitive approaches. Advancements in this area routinely employ labels, label free, nanoparticles, multifunctional matrices, carbon nanotubes and other methods to meet the requirements of its own application. However, despite increasing affinity ceilings for conventional biosensors, the field draws back in meeting specifically important demands, such as long-term stability, ultrasensitivity, rapid detection, extreme selectivity, strong biological base, calibration, in vivo measurements, regeneration, satisfactory performance and ease of production. Nevertheless, recent efforts through this line have produced novel high-tech nanosensing systems such as ‘aptamers’ and ‘phages’ which exhibit high-throughput sensing. Aptamers and phages are powerful tools that excel over antibodies in sensibility, stability, multi-detection, in vivo measurements and regeneration. Phages are superior in stability, screening for affinity-based target molecules ranging from small to proteins and even cells, and easy production. In this review, we focus mainly on recent developments in affinity-based biosensors such as immunosensors, DNA sensors, emphasizing aptasensors and phage-based biosensors basing on novel electrochemical, optical and mass sensitive detection techniques. We also address enzyme inhibition-based biosensors and the current problems associated with the above sensors and their future perspectives.

  14. Semiempirical Theories of the Affinities of Negative Atomic Ions

    NASA Technical Reports Server (NTRS)

    Edie, John W.

    1961-01-01

    The determination of the electron affinities of negative atomic ions by means of direct experimental investigation is limited. To supplement the meager experimental results, several semiempirical theories have been advanced. One commonly used technique involves extrapolating the electron affinities along the isoelectronic sequences, The most recent of these extrapolations Is studied by extending the method to Include one more member of the isoelectronic sequence, When the results show that this extension does not increase the accuracy of the calculations, several possible explanations for this situation are explored. A different approach to the problem is suggested by the regularities appearing in the electron affinities. Noting that the regular linear pattern that exists for the ionization potentials of the p electrons as a function of Z, repeats itself for different degrees of ionization q, the slopes and intercepts of these curves are extrapolated to the case of the negative Ion. The method is placed on a theoretical basis by calculating the Slater parameters as functions of q and n, the number of equivalent p-electrons. These functions are no more than quadratic in q and n. The electron affinities are calculated by extending the linear relations that exist for the neutral atoms and positive ions to the negative ions. The extrapolated. slopes are apparently correct, but the intercepts must be slightly altered to agree with experiment. For this purpose one or two experimental affinities (depending on the extrapolation method) are used in each of the two short periods. The two extrapolation methods used are: (A) an isoelectronic sequence extrapolation of the linear pattern as such; (B) the same extrapolation of a linearization of this pattern (configuration centers) combined with an extrapolation of the other terms of the ground configurations. The latter method Is preferable, since it requires only experimental point for each period. The results agree within

  15. Affine Covariant Features for Fisheye Distortion Local Modelling.

    PubMed

    Furnari, Antonino; Farinella, Giovanni; Bruna, Arcangelo; Battiato, Sebastiano

    2016-11-10

    Perspective cameras are the most popular imaging sensors used in Computer Vision. However, many application fields including automotive, surveillance and robotics, require the use of wide angle cameras (e.g., fisheye), which allow to acquire a larger portion of the scene using a single device at the cost of the introduction of noticeable radial distortion in the images. Affine covariant feature detectors have proven successful in a variety of Computer Vision applications including object recognition, image registration and visual search. Moreover, their robustness to a series of variabilities related to both the scene and the image acquisition process has been thoroughly studied in the literature. In this paper, we investigate their effectiveness on fisheye images providing both theoretical and experimental analyses. As theoretical outcome, we show that the inherently non-linear radial distortion can be locally approximated by linear functions with a reasonably small error. The experimental analysis builds on Mikolajczyk's benchmark to assess the robustness of three popular affine region detectors (i.e., Maximally Stable Extremal Regions (MSER), Harris and Hessian affine region detectors), with respect to different variabilities as well as to radial distortion. To support the evaluations, we rely on the Oxford dataset and introduce a novel benchmark dataset comprising 50 images depicting different scene categories. Experiments are carried out on rectilinear images to which radial distortion is artificially added, and on real-world images acquired using fisheye lenses. Our analysis points out that affine region detectors can be effectively employed directly on fisheye images and that the radial distortion is locally modelled as an additional affine variability.

  16. Imaging mass spectrometry in microbiology

    PubMed Central

    Watrous, Jeramie D.; Dorrestein, Pieter C.

    2013-01-01

    Mass spectrometry tools which allow for the 2-D visualization of the distribution of trace metals, metabolites, surface lipids, peptides and proteins directly from biological samples without the need for chemical tagging or antibodies are becoming increasingly useful for microbiology applications. These tools, comprised of different imaging mass spectrometry techniques, are ushering in an exciting new era of discovery by allowing for the generation of chemical hypotheses based on of the spatial mapping of atoms and molecules that can correlate to or transcend observed phenotypes. In this review, we explore the wide range of imaging mass spectrometry techniques available to microbiologists and describe their unique applications to microbiology with respect to the types of microbiology samples to be investigated. PMID:21822293

  17. Symposium on accelerator mass spectrometry

    SciTech Connect

    1981-01-01

    The area of accelerator mass spectrometry has expanded considerably over the past few years and established itself as an independent and interdisciplinary research field. Three years have passed since the first meeting was held at Rochester. A Symposium on Accelerator Mass Spectrometry was held at Argonne on May 11-13, 1981. In attendance were 96 scientists of whom 26 were from outside the United States. The present proceedings document the program and excitement of the field. Papers are arranged according to the original program. A few papers not presented at the meeting have been added to complete the information on the status of accelerator mass spectrometry. Individual papers were prepared separately for the data base.

  18. Mass spectrometry. [in organic chemistry

    NASA Technical Reports Server (NTRS)

    Burlingame, A. L.; Shackleton, C. H. L.; Howe, I.; Chizhov, O. S.

    1978-01-01

    A review of mass spectrometry in organic chemistry is given, dealing with advances in instrumentation and computer techniques, selected topics in gas-phase ion chemistry, and applications in such fields as biomedicine, natural-product studies, and environmental pollution analysis. Innovative techniques and instrumentation are discussed, along with chromatographic-mass spectrometric on-line computer techniques, mass spectral interpretation and management techniques, and such topics in gas-phase ion chemistry as electron-impact ionization and decomposition, photoionization, field ionization and desorption, high-pressure mass spectrometry, ion cyclotron resonance, and isomerization reactions of organic ions. Applications of mass spectrometry are examined with respect to bio-oligomers and their constituents, biomedically important substances, microbiology, environmental organic analysis, and organic geochemistry.

  19. A fluorescence nanosensor for glycoproteins with activity based on the molecularly imprinted spatial structure of the target and boronate affinity.

    PubMed

    Zhang, Wei; Liu, Wei; Li, Ping; Xiao, Haibin; Wang, Hui; Tang, Bo

    2014-11-10

    Glycoproteins are closely associated with the occurrence of diverse diseases, and they have been used as biomarkers and therapeutic targets in clinical diagnostics. Currently, mass spectrometry has proven to be a powerful tool for glycoprotein analysis, but it is almost impossible to directly identify glycoproteins without the preparation and pretreatment of samples. Furthermore, biological samples, especially proteins, are damaged by this process. Herein, we describe a novel fluorescence nanosensor based on a molecularly imprinted spatial structure and boronate affinity that is well-suited for monitoring glycoproteins selectively. Results showed that the recognition performance of the nanosensor for glycoproteins was regulated by controlling the pH value and temperature. Moreover, the nanosensor was successfully applied to the detection of HRP in biological fluids. This study provides a facile and efficient fluorescence tool for glycoprotein detection in clinical diagnostics.

  20. A novel affinity-based method for the isolation of highly purified extracellular vesicles

    PubMed Central

    Nakai, Wataru; Yoshida, Takeshi; Diez, Diego; Miyatake, Yuji; Nishibu, Takahiro; Imawaka, Naoko; Naruse, Ken; Sadamura, Yoshifusa; Hanayama, Rikinari

    2016-01-01

    Extracellular vesicles (EVs) such as exosomes and microvesicles serve as messengers of intercellular network, allowing exchange of cellular components between cells. EVs carry lipids, proteins, and RNAs derived from their producing cells, and have potential as biomarkers specific to cell types and even cellular states. However, conventional methods (such as ultracentrifugation or polymeric precipitation) for isolating EVs have disadvantages regarding purity and feasibility. Here, we have developed a novel method for EV purification by using Tim4 protein, which specifically binds the phosphatidylserine displayed on the surface of EVs. Because the binding is Ca2+-dependent, intact EVs can be easily released from Tim4 by adding Ca2+ chelators. Tim4 purification, which we have applied to cell conditioned media and biofluids, is capable of yielding EVs of a higher purity than those obtained using conventional methods. The lower contamination found in Tim4-purified EV preparations allows more EV-specific proteins to be detected by mass spectrometry, enabling better characterization and quantification of different EV populations’ proteomes. Tim4 protein can also be used as a powerful tool for quantification of EVs in both ELISA and flow cytometry formats. Thus, the affinity of Tim4 for EVs will find abundant applications in EV studies. PMID:27659060

  1. Direct isolation of specific RNA-interacting proteins using a novel affinity medium.

    PubMed

    Liu, Ding-Gan; Sun, Li

    2005-08-26

    Isolation of proteins that specifically interact with a given RNA or RNA regulation element is essential for studies on the molecular mechanisms of gene expression. Here, a novel method for direct isolation of such interacting proteins is described. It uses an affinity medium that consists of an interacting RNA with an artificially added 'tail', which is annealed to one end of a DNA 'arm', the other end of which is fixed covalently on the surface of aminosilanized glass powder. Thus the RNA itself is fully suspending, facilitating its interactions with proteins in its natural conformation. The proteins bound on the interacting RNA are eluted and subjected to SDS-PAGE, and the Coomassie-stained protein bands are cut and subjected to mass spectrometry (MS) analysis. Using this method, three proteins specifically interacting with the C/EBPbeta 3'-untranslated region (3'-UTR) RNA were isolated and identified. This method is simple and convenient, and the DNA-glass powder medium can be used repeatedly.

  2. High Confidence Fission Yeast SUMO Conjugates Identified by Tandem Denaturing Affinity Purification.

    PubMed

    Nie, Minghua; Vashisht, Ajay A; Wohlschlegel, James A; Boddy, Michael N

    2015-09-25

    Covalent attachment of the small ubiquitin-like modifier (SUMO) to key targets in the proteome critically regulates the evolutionarily conserved processes of cell cycle control, transcription, DNA replication and maintenance of genome stability. The proteome-wide identification of SUMO conjugates in budding yeast has been invaluable in helping to define roles of SUMO in these processes. Like budding yeast, fission yeast is an important and popular model organism; however, the fission yeast Schizosaccharomyces pombe community currently lacks proteome-wide knowledge of SUMO pathway targets. To begin to address this deficiency, we adapted and used a highly stringent Tandem Denaturing Affinity Purification (TDAP) method, coupled with mass spectrometry, to identify fission yeast SUMO conjugates. Comparison of our data with that compiled in budding yeast reveals conservation of SUMO target enrichment in nuclear and chromatin-associated processes. Moreover, the SUMO "cloud" phenomenon, whereby multiple components of a single protein complex are SUMOylated, is also conserved. Overall, SUMO TDAP provides both a key resource of high confidence SUMO-modified target proteins in fission yeast, and a robust method for future analyses of SUMO function.

  3. Fabrication of enzyme-immobilized halloysite nanotubes for affinity enrichment of lipase inhibitors from complex mixtures.

    PubMed

    Wang, Haibo; Zhao, Xiaoping; Wang, Shufang; Tao, Shan; Ai, Ni; Wang, Yi

    2015-05-01

    Lipase is the key enzyme for catalyzing triglyceride hydrolysis in vivo, and lipase inhibitors have been used in the management of obesity. We present the first report on the use of lipase-adsorbed halloysite nanotubes as an efficient medium for the selective enrichment of lipase inhibitors from natural products. A simple and rapid approach was proposed to fabricate lipase-adsorbed nanotubes through electrostatic interaction. Results showed that more than 85% lipase was adsorbed into nanotubes in 90 min, and approximately 80% of the catalytic activity was maintained compared with free lipase. The specificity and reproducibility of the proposed approach were validated by screening a known lipase inhibitor (i.e., orlistat) from a mixture that contains active and inactive compounds. Moreover, we applied this approach with high performance liquid chromatography-mass spectrometry technique to screen lipase inhibitors from the Magnoliae cortex extract, a medicinal plant used for treating obesity. Two novel biphenyl-type natural lipase inhibitors magnotriol A and magnaldehyde B were identified, and their IC50 values were determined as 213.03 and 96.96 μM, respectively. The ligand-enzyme interactions of magnaldehyde B were further investigated by molecular docking. Our findings proved that enzyme-adsorbed nanotube could be used as a feasible and selective affinity medium for the rapid screening of enzyme inhibitors from complex mixtures.

  4. Improved tandem affinity purification tag and methods for isolation of protein heterocomplexes from plants.

    PubMed

    Rohila, Jai S; Chen, Mei; Cerny, Ronald; Fromm, Michael E

    2004-04-01

    A synthetic gene encoding the tandem affinity purification (TAP) tag has been constructed, and the TAP tag assayed for its effects on expression levels and subcellular localization by fusion to green fluorescent protein (GFP) as well as for its effects on steroid-dependent translocation to the nucleus and transcription when fused to a hybrid glucocorticoid receptor. A nuclear localization signal (NLS) was detected in the calmodulin-binding protein (CBP) domain and removed by mutation to improve the usefulness of the TAP tag. Additionally, purification improvements were made, including inhibition of a co-purifying protease, and adding a protein cross-linking step to increase the recovery of interacting proteins. The improved synthetic TAP tag gene and methods were used to isolate proteins interacting with the hybrid glucocorticoid receptor and to identify them by mass spectrometry. The two proteins identified, HSP70 and HSP90, are known to interact with the glucocorticoid receptor in vivo in mammalian cells and in vitro in plants.

  5. PHOS-Select Iron Affinity beads enrich peptides for detection of organophosphorus adducts on albumin

    PubMed Central

    Jiang, Wei; Dubrovskii, Yaroslav A; Podolskaya, Ekaterina P; Murashko, Ekaterina A; Babakov, Vladimir; Nachon, Florian; Masson, Patrick; Schopfer, Lawrence M; Lockridge, Oksana

    2013-01-01

    Albumin is covalently modified by organophosphorus toxicants (OP) on tyrosine 411, but less than 1% of albumin is modified in humans by lethal OP doses that inhibit 95% of plasma butyrylcholinesterase. A method that enriches OP-modified albumin peptides could aid analysis of low dose exposures. Soman or chlorpyrifos oxon treated human plasma was digested with pepsin. Albumin peptides were enriched by binding to Fe3+ beads at pH 11 and eluted with pH 2.6 buffer. Similarly, mouse and guinea pig albumin modified by chlorpyrifos oxon were digested with pepsin and enriched by binding to Fe3+ beads. Peptides were identified by MALDI-TOF/TOF mass spectrometry. PHOS-select Iron Affinity beads specifically enriched albumin peptides VRY411TKKVPQVST and LVRY411TKKVPQVST in a pepsin digest of human plasma. The unmodified as well as OP-modified peptides bound to the beads. The binding capacity of 500 μl beads was the pepsin digest of 2.1 μL human plasma. The limit of detection was 0.2% of OP-modified albumin peptide in 0.43 μL plasma. Enrichment of OP-modified albumin peptides by binding to Fe3+ beads is a method with potential application to diagnosis of OP pesticide and nerve agent exposure in humans, mice, and guinea pigs. PMID:24187955

  6. Binding site on human immunoglobulin G for the affinity ligand HWRGWV

    PubMed Central

    Yang, Haiou; Gurgel, Patrick V.; Williams, D. Keith; Bobay, Benjamin G.; Cavanagh, John; Muddiman, David C.; Carbonell, Ruben G.

    2014-01-01

    Affinity ligand HWRGWV has demonstrated the ability to isolate human immunoglobulin G (hIgG) from mammalian cell culture media. The ligand specifically binds hIgG through its Fc portion. This work shows that deglycosylation of hIgG has no influence on its binding to the HWRGWV ligand and the ligand does not compete with Protein A or Protein G in binding hIgG. It is suggested by the mass spectrometry (MS) data and docking simulation that HWRGWV binds to the pFc portion of hIgG and interacts with the amino acids in the loop Ser383–Asn389 (SNGQPEN) located in the CH3 domain. Subsequent modeling has suggested a possible three-dimensional minimized solution structure for the interaction of hIgG and the HWRGWV ligand. The results support the fact that a peptide as small as a hexamer can have specific interactions with large proteins such as hIgG. PMID:20049844

  7. Purification of prenylated proteins by affinity chromatography on cyclodextrin-modified agarose

    PubMed Central

    Chung, Jinhwa A.; Wollack, James W.; Hovlid, Marisa L.; Okesli, Ayse; Chen, Yan; Mueller, Joachim D.; Distefano, Mark D.; Taton, T. Andrew

    2009-01-01

    Although protein prenylation is widely studied, there are few good methods for isolating prenylated proteins from their non-prenylated relatives. We report that crosslinked agarose (e.g., Sepharose) chromatography media that has been chemically functionalized with β-cyclodextrin (β-CD) is extremely effective in affinity chromatography of prenylated proteins. In this study, a variety of proteins with C-terminal prenylation target (“CAAX box”) sequences were enzymatically prenylated in vitro with natural and non-natural prenyl diphosphate substrates. The prenylated protein products could then be isolated from starting materials by gravity chromatography or fast protein liquid chromatography (FPLC) on a β-CD-Sepharose column. One particular prenylation reaction—farnesylation of a mCherry-CAAX fusion construct—was studied in detail. In this case, purified farnesylated product was unambiguously identified by electrospray mass spectrometry. In addition, when mCherry-CAAX was prenylated with a non-natural, functional isoprenoid substrate, the functional group was maintained by chromatography on β-CD-Sepharose, such that the resulting protein could be selectively bound at its C terminus to complementary functionality on a solid substrate. Finally, β-CD-Sepharose FPLC was used to isolate prenylated mCherry-CAAX from crude HeLa cell lysate, as a model for purifying prenylated proteins from cell extracts. We propose that this method could be generally useful to the community of researchers studying protein prenylation. PMID:18834849

  8. Specific interaction between negative atmospheric ions and organic compounds in atmospheric pressure corona discharge ionization mass spectrometry.

    PubMed

    Sekimoto, Kanako; Sakai, Mami; Takayama, Mitsuo

    2012-06-01

    The interaction between negative atmospheric ions and various types of organic compounds were investigated using atmospheric pressure corona discharge ionization (APCDI) mass spectrometry. Atmospheric negative ions such as O(2)(-), HCO(3)(-), COO(-)(COOH), NO(2)(-), NO(3)(-), and NO(3)(-)(HNO(3)) having different proton affinities served as the reactant ions for analyte ionization in APCDI in negative-ion mode. The individual atmospheric ions specifically ionized aliphatic and aromatic compounds with various functional groups as atmospheric ion adducts and deprotonated analytes. The formation of the atmospheric ion adducts under certain discharge conditions is most likely attributable to the affinity between the analyte and atmospheric ion and the concentration of the atmospheric ion produced under these conditions. The deprotonated analytes, in contrast, were generated from the adducts of the atmospheric ions with higher proton affinity attributable to efficient proton abstraction from the analyte by the atmospheric ion.

  9. Mass spectrometry guided structural biology.

    PubMed

    Liko, Idlir; Allison, Timothy M; Hopper, Jonathan Ts; Robinson, Carol V

    2016-10-01

    With the convergence of breakthroughs in structural biology, specifically breaking the resolution barriers in cryo-electron microscopy and with continuing developments in crystallography, novel interfaces with other biophysical methods are emerging. Here we consider how mass spectrometry can inform these techniques by providing unambiguous definition of subunit stoichiometry. Moreover recent developments that increase mass spectral resolution enable molecular details to be ascribed to unassigned density within high-resolution maps of membrane and soluble protein complexes. Importantly we also show how developments in mass spectrometry can define optimal solution conditions to guide downstream structure determination, particularly of challenging biomolecules that refuse to crystallise.

  10. Experimental allergic encephalomyelitis (EAE) in mice selectively bred to produce high affinity (HA) or low affinity (LA) antibody responses.

    PubMed Central

    Devey, M E; Major, P J; Bleasdale-Barr, K M; Holland, G P; Dal Canto, M C; Paterson, P Y

    1990-01-01

    Induction of experimental allergic encephalomyelitis (EAE) in mice genetically selected to produce either high affinity (HA) or low affinity (LA) antibody responses has revealed significant differences in disease susceptibility between the two lines. HA mice were highly susceptible to EAE following subcutaneous sensitization to mouse central nervous system (CNS) tissue emulsified in Freund's complete adjuvant (FCA). Furthermore, of HA mice surviving acute EAE, up to 93% subsequently developed chronic relapsing disease (CREAE) characterized by variable demyelinating inflammatory changes within the spinal cord. In contrast, LA mice, despite having a major histocompatability complex (MHC) haplotype associated with susceptibility to EAE, were highly resistant to the disease and showed no signs of CREAE when observed for up to 100 days post-sensitization. Antibodies to myelin basic protein (MBP) were detected in both lines but rising titres of high functional affinity antibodies were only seen in HA mice. These HA and LA lines of mice provide a new approach to the study of EAE and, in particular, the role of antibody and antibody affinity in the chronic relapsing form of the disease. Images Figure 2 PMID:2335373

  11. PDZ Affinity Chromatography: A general method for affinity purification of proteins based on PDZ domains and their ligands

    PubMed Central

    Walkup, Ward G.; Kennedy, Mary B.

    2014-01-01

    PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ~ 90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ-domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and β-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins. PMID:24607360

  12. PDZ affinity chromatography: a general method for affinity purification of proteins based on PDZ domains and their ligands.

    PubMed

    Walkup, Ward G; Kennedy, Mary B

    2014-06-01

    PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ∼90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and β-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins.

  13. Phosphoproteomics by mass spectrometry and classical protein chemistry approaches.

    PubMed

    Salih, Erdjan

    2005-01-01

    The general fields of biological sciences have seen phenomenal transformations in the past two decades at the level of data acquisition, understanding biological processes, and technological developments. Those advances have been made partly because of the advent of molecular biology techniques (which led to genomics) coupled to the advances made in mass spectrometry (MS) to provide the current capabilities and developments in proteomics. However, our current knowledge that approximately 30,000 human genes may code for up to 1 million or more proteins disengage the interface between the genome sequence database algorithms and MS to generate a major interest in independent de novo MS/MS sequence determination. Significant progress has been made in this area through procedures to covalently modify peptide N- and C-terminal amino-acids by sulfonation and guanidination to permit rapid de novo sequence determination by MS/MS analysis. A number of strategies that have been developed to perform qualitative and quantitative proteomics range from 2D-gel electrophoresis, affinity tag reagents, and stable-isotope labeling. Those procedures, combined with MS/MS peptide sequence analysis at the subpicomole level, permit the rapid and effective identification and quantification of a large number of proteins within a given biological sample. The identification of proteins per se, however, is not always sufficient to interpret biological function because many of the naturally occurring proteins are post-translationally modified. One such modification is protein phosphorylation, which regulates a large array of cellular biochemical pathways of the biological system. Traditionally, the study of phosphoprotein structure-function relationships involved classical protein chemistry approaches that required protein purification, peptide mapping, and the identification of the phosphorylated peptide regions and sites by N-terminal sequence analysis. Recent advances made in mass

  14. Affinity learning with diffusion on tensor product graph.

    PubMed

    Yang, Xingwei; Prasad, Lakshman; Latecki, Longin Jan

    2013-01-01

    In many applications, we are given a finite set of data points sampled from a data manifold and represented as a graph with edge weights determined by pairwise similarities of the samples. Often the pairwise similarities (which are also called affinities) are unreliable due to noise or due to intrinsic difficulties in estimating similarity values of the samples. As observed in several recent approaches, more reliable similarities can be obtained if the original similarities are diffused in the context of other data points, where the context of each point is a set of points most similar to it. Compared to the existing methods, our approach differs in two main aspects. First, instead of diffusing the similarity information on the original graph, we propose to utilize the tensor product graph (TPG) obtained by the tensor product of the original graph with itself. Since TPG takes into account higher order information, it is not a surprise that we obtain more reliable similarities. However, it comes at the price of higher order computational complexity and storage requirement. The key contribution of the proposed approach is that the information propagation on TPG can be computed with the same computational complexity and the same amount of storage as the propagation on the original graph. We prove that a graph diffusion process on TPG is equivalent to a novel iterative algorithm on the original graph, which is guaranteed to converge. After its convergence we obtain new edge weights that can be interpreted as new, learned affinities. We stress that the affinities are learned in an unsupervised setting. We illustrate the benefits of the proposed approach for data manifolds composed of shapes, images, and image patches on two very different tasks of image retrieval and image segmentation. With learned affinities, we achieve the bull's eye retrieval score of 99.99 percent on the MPEG-7 shape dataset, which is much higher than the state-of-the-art algorithms. When the data

  15. Purification of papain by metal affinity partitioning in aqueous two-phase polyethylene glycol/sodium sulfate systems.

    PubMed

    Jiang, Zhi-Guo; Zhang, Hai-De; Wang, Wei-Tao

    2015-05-01

    A simple and inexpensive aqueous two-phase affinity partitioning system using metal ligands was introduced to improve the selectivity of commercial papain extraction. Polyethylene glycol 4000 was first activated using epichlorohydrin, then it was covalently linked to iminodiacetic acid. Finally, the specific metal ligand Cu(2+) was attached to the polyethylene glycol-iminodiacetic acid. The chelated Cu(2+) content was measured by atomic absorption spectrometry as 0.88 mol/mol (polyethylene glycol). The effects on the purification at different conditions, including polyethylene glycol molecular weight (2000, 4000, and 6000), concentration of phase-forming components (polyethylene glycol 12-20% w/w and sodium sulfate 12-20%, w/w), metal ligand type, and concentration, system pH and the commercial papain loading on papain extraction, were systematically studied. Under optimum conditions of the system, i.e. 18% w/w sodium sulfate, 18% w/w polyethylene glycol 4000, 1% w/w polyethylene glycol-iminodiacetic acid-Cu(2+) and pH 7, a maximum yield of 90.3% and a degree of purification of 3.6-fold were obtained. Compared to aqueous two phase extraction without ligands, affinity partitioning was found to be an effective technique for the purification of commercial papain with higher extraction efficiency and degree of purification.

  16. An in vivo imaging-based assay for detecting protein interactions over a wide range of binding affinities

    SciTech Connect

    Fowlkes, Jason Davidson; Owens, Elizabeth T; Standaert, Robert F; Pelletier, Dale A; Hurst, Gregory {Greg} B; Doktycz, Mitchel John; Morrell-Falvey, Jennifer L; Billings, Amanda N

    2009-01-01

    Identifying and characterizing protein interactions are fundamental steps towards understanding and modeling biological networks. Methods that detect protein interactions in intact cells rather than buffered solutions are likely more relevant to natural systems since molecular crowding events in the cytosol can influence the diffusion and reactivity of individual proteins. One in vivo, imaging-based method relies on the co-localization of two proteins of interest fused to DivIVA, a cell division protein from Bacillus subtilis, and green fluorescent protein (GFP). We have modified this imaging-based assay to facilitate rapid cloning by constructing new vectors encoding N- and C-terminal DivIVA or GFP molecular tag fusions based on site-specific recombination technology. The sensitivity of the assay was defined using a well-characterized protein interaction system involving the eukaryotic nuclear import receptor subunit, Importin (Imp ) and variant nuclear localization signals (NLS) representing a range of binding affinities. These data demonstrate that the modified co-localization assay is sensitive enough to detect protein interactions with Kd values that span over four orders of magnitude (1nM to 15 M). Lastly, this assay was used to confirm numerous protein interactions identified from mass spectrometry-based analyses of affinity isolates as part of an interactome mapping project in Rhodopseudomonas palustris

  17. Ultra sensitive affinity chromatography on avidin-functionalized PMMA microchip for low abundant post-translational modified protein enrichment.

    PubMed

    Xia, Hui; Murray, Kermit; Soper, Steven; Feng, June

    2012-02-01

    Post-translational modifications (PTM) of proteins play essential roles in cellular physiology and disease. The identification of protein substrates and detection of modification site helps understand PTM-mediated regulation in essential biological pathways and functions in various diseases. However, PTM proteins are typically present only at trace levels, making them difficult to identify in mass spectrometry based proteomics. In this paper, we report a novel and sensitive affinity chromatography on the avidin-functionalized poly(methyl methacrylate) (PMMA) microchip for enrichment of nanogram (ng) amount of PTMs. The chemical modification of poly(methyl methacrylate) (PMMA) surfaces yield avidin-terminated PMMA surfaces after UV radiation and consecutive EDC mediated coupling (amide reaction). This functionalized PMMA micro-device was developed to identify and specifically trap biotinylated PTM proteins of low abundance from complex protein mixture. Here we selected carbonylated protein as a representative PTM to illustrate the wide application of this affinity microchip for any PTMs converted into a tractable tag after derivatization. The surface topography, surface functional group mapping and elemental composition changes after each modification step of the treatment process were systematically measured qualitatively and quantitatively by atomic force microscopy, X-ray photoelectron spectroscopy and fluorescence microscopy. Quantitative study of biotinlated carbonylated protein capture recovery and elution efficiency of the device was also studied. We also envision that this subproteome enrichment micro-device can be assembled with other lab-on-a-chip components for follow-up protein analysis.

  18. Applications of a catch and release electrospray ionization mass spectrometry assay for carbohydrate library screening.

    PubMed

    El-Hawiet, Amr; Shoemaker, Glen K; Daneshfar, Rambod; Kitova, Elena N; Klassen, John S

    2012-01-03

    Applications of a catch and release electrospray ionization mass spectrometry (CaR-ESI-MS) assay for screening carbohydrate libraries against target proteins are described. Direct ESI-MS measurements were performed on solutions containing a target protein (a single chain antibody, an antigen binding fragment, or a fragment of a bacterial toxin) and a library of carbohydrates containing multiple specific ligands with affinities in the 10(3) to 10(6) M(-1) range. Ligands with moderate affinity (10(4) to 10(6) M(-1)) were successfully detected from mixtures containing >200 carbohydrates (at concentrations as low as 0.25 μM each). Additionally, the absolute affinities were estimated from the abundance of free and ligand-bound protein ions determined from the ESI mass spectrum. Multiple low affinity ligands (~10(3) M(-1)) were successfully detected in mixtures containing >20 carbohydrates (at concentrations of ~10 μM each). However, identification of specific interactions required the use of the reference protein method to correct the mass spectrum for the occurrence of nonspecific carbohydrate-protein binding during the ESI process. The release of the carbohydrate ligands, as ions, was successfully demonstrated using collision-induced dissociation performed on the deprotonated ions of the protein-carbohydrate complexes. The use of ion mobility separation, performed on deprotonated carbohydrate ions following their release from the complex, allowed for the positive identification of isomeric ligands.

  19. Mass Spectrometry for the Masses

    ERIC Educational Resources Information Center

    Persinger, Jared D.; Hoops, Geoffrey, C.; Samide, Michael J.

    2004-01-01

    A simple, qualitative experiment is developed for implementation, where the gas chromatography-mass spectrometry (GC-MS) plays an important role, into the laboratory curriculum of a chemistry course designed for nonscience majors. This laboratory experiment is well suited for the students as it helps them to determine the validity of their…

  20. Ultraviolet and Light Absorption Spectrometry.

    ERIC Educational Resources Information Center

    Hargis, L. G.; Howell, J. A.

    1984-01-01

    Reviews developments in ultraviolet and light absorption spectrometry from December 1981 through November 1983, focusing on the chemistry involved in developing suitable reagents, absorbing systems, and methods of determination, and on physical aspects of the procedures. Includes lists of spectrophotometric methods for metals, non-metals, and…

  1. Linear electric field mass spectrometry

    DOEpatents

    McComas, D.J.; Nordholt, J.E.

    1992-12-01

    A mass spectrometer and methods for mass spectrometry are described. The apparatus is compact and of low weight and has a low power requirement, making it suitable for use on a space satellite and as a portable detector for the presence of substances. High mass resolution measurements are made by timing ions moving through a gridless cylindrically symmetric linear electric field. 8 figs.

  2. Linear electric field mass spectrometry

    DOEpatents

    McComas, David J.; Nordholt, Jane E.

    1992-01-01

    A mass spectrometer and methods for mass spectrometry. The apparatus is compact and of low weight and has a low power requirement, making it suitable for use on a space satellite and as a portable detector for the presence of substances. High mass resolution measurements are made by timing ions moving through a gridless cylindrically symmetric linear electric field.

  3. "EMERGING" POLLUTANTS, MASS SPECTROMETRY, AND ...

    EPA Pesticide Factsheets

    A foundation for Environmental Science - Mass Spectrometry: Historically fundamental to amassing our understanding of environmental processes and chemical pollution is the realm of mass spectrometry - the mainstay of analytical chemistry - the workhorse that supplies much of the definitive data that environmental scientists rely upon for identifying the molecular compositions (and ultimately the structures) of chemicals. This is not to ignore the complementary, critical roles played by the adjunct practices of sample enrichment (via any of various means of selective extraction) and analyte separation (via the myriad forms of chromatography and electrophoresis).While the power of mass spectrometry has long been highly visible to the practicing environmental chemist, it borders on continued obscurity to the lay public and most non-chemists. Even though mass spectrometry has played a long, historic (and largely invisible) role in establishing or undergirdidng our existing knowledge about environmental processes and pollution, what recognition it does enjoy is usually relegated to that of a tool. It is ususally the relevance of ssignificance of the knowledge acquired from the application of the tool that has ultimate meaning to the public and science at large - not how the knowledge was acquired. The research focused on in the subtasks is the development and application of state-of the-art technologies to meet the needs of the public, Office of Water, and ORD in

  4. Synthesis and application of novel phenylboronate affinity materials based on organic polymer particles for selective trapping of glycoproteins.

    PubMed

    Preinerstorfer, Beatrix; Lämmerhofer, Michael; Lindner, Wolfgang

    2009-05-01

    We report on synthesis concepts for the fabrication of various novel phenylboronate affinity materials based on polymethacrylate epoxy beads (Fractogel EMD Epoxy (M) 40-90 microm) and the testing of these functionalized polymer particles for selective trapping of a glycoprotein from a standard mixture containing a glycosylated and a nonglycosylated protein. Two inherently different approaches for the functionalization of the bare beads with boronate groups have been elucidated. In the first, the epoxy residues of the polymer particles were converted into reactive thiol groups which were subsequently used as anchor moieties for the immobilization of 4-vinylphenylboronic acid by radical addition or radical polymerization reaction. Three different ways for the generation of sulfhydryl groups have been examined leading to materials with distinct linker chemistries. In the second and more straightforward approach, the epoxy groups were reacted with 4-mercaptophenylboronic acid. The novel materials were thoroughly characterized by (i) quantitation of the sulfur content by elemental analysis, (ii) reactive sulfhydryls were determined in a photospectrometric assay, (iii) boron content was measured by inductively coupled plasma-atomic emission spectrometry, and (iv) the amount of reactive boronate groups was evaluated in a fast binding assay employing adenosine as test compound. A maximum concentration of 1.2 mmol boronate groups per gram dry beads could be achieved by the presented synthesis routes. Employing the novel phenylboronate affinity materials in capture and release experiments in the batch mode, a standard glycoprotein, viz. transferrin (Tf) from human serum was separated from a nonglycosylated protein, BSA. A commercial boronate affinity material based on 3-aminophenylboronic acid modified agarose gel was employed as reference material and was found to perform significantly worse compared to the herein presented novel polymethacrylate particles.

  5. Characterization of naturally-occurring and modified fullerenes by Fourier transform mass spectrometry

    SciTech Connect

    Hettich, R.L.; Jin, C.; Compton, R.N. ); Buseck, P.R.; Tsipursky, S.J. )

    1993-10-10

    Fourier transform mass spectrometry (FTMS) employing both laser desorption/ionization and thermal desorption/electron ionization is useful for the detection and structural characterization of fullerenes and chemically-modified fullerenes. Examination of a carbon-rich shungite rock sample from Russia by transmission electron microscopy and FTMS provided evidence of naturally-occurring fullerenes. Ion-molecule reactions can be studied with FTMS to investigate the electron affinities of modified fullerenes. By monitoring charge exchange reactions, the electron affinities of C[sub 60]F[sub x] (x=44,46) and C[sub 70]F[sub y] (y=52,54) were found to be substantially higher than the values for the parent fullerenes.

  6. Screening Anti-Cancer Drugs against Tubulin using Catch-and-Release Electrospray Ionization Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Rezaei Darestani, Reza; Winter, Philip; Kitova, Elena N.; Tuszynski, Jack A.; Klassen, John S.

    2016-05-01

    Tubulin, which is the building block of microtubules, plays an important role in cell division. This critical role makes tubulin an attractive target for the development of chemotherapeutic drugs to treat cancer. Currently, there is no general binding assay for tubulin-drug interactions. The present work describes the application of the catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay to investigate the binding of colchicinoid drugs to αβ-tubulin dimers extracted from porcine brain. Proof-of-concept experiments using positive (ligands with known affinities) and negative (non-binders) controls were performed to establish the reliability of the assay. The assay was then used to screen a library of seven colchicinoid analogues to test their binding to tubulin and to rank their affinities.

  7. Model updating based on an affine scaling interior optimization algorithm

    NASA Astrophysics Data System (ADS)

    Zhang, Y. X.; Jia, C. X.; Li, Jian; Spencer, B. F.

    2013-11-01

    Finite element model updating is usually considered as an optimization process. Affine scaling interior algorithms are powerful optimization algorithms that have been developed over the past few years. A new finite element model updating method based on an affine scaling interior algorithm and a minimization of modal residuals is proposed in this article, and a general finite element model updating program is developed based on the proposed method. The performance of the proposed method is studied through numerical simulation and experimental investigation using the developed program. The results of the numerical simulation verified the validity of the method. Subsequently, the natural frequencies obtained experimentally from a three-dimensional truss model were used to update a finite element model using the developed program. After updating, the natural frequencies of the truss and finite element model matched well.

  8. Learning factorizations in estimation of distribution algorithms using affinity propagation.

    PubMed

    Santana, Roberto; Larrañaga, Pedro; Lozano, José A

    2010-01-01

    Estimation of distribution algorithms (EDAs) that use marginal product model factorizations have been widely applied to a broad range of mainly binary optimization problems. In this paper, we introduce the affinity propagation EDA (AffEDA) which learns a marginal product model by clustering a matrix of mutual information learned from the data using a very efficient message-passing algorithm known as affinity propagation. The introduced algorithm is tested on a set of binary and nonbinary decomposable functions and using a hard combinatorial class of problem known as the HP protein model. The results show that the algorithm is a very efficient alternative to other EDAs that use marginal product model factorizations such as the extended compact genetic algorithm (ECGA) and improves the quality of the results achieved by ECGA when the cardinality of the variables is increased.

  9. Affinity-Driven Immobilization of Proteins to Hematite Nanoparticles.

    PubMed

    Zare-Eelanjegh, Elaheh; Bora, Debajeet K; Rupper, Patrick; Schrantz, Krisztina; Thöny-Meyer, Linda; Maniura-Weber, Katharina; Richter, Michael; Faccio, Greta

    2016-08-10

    Functional nanoparticles are valuable materials for energy production, bioelectronics, and diagnostic devices. The combination of biomolecules with nanosized material produces a new hybrid material with properties that can exceed the ones of the single components. Hematite is a widely available material that has found application in various sectors such as in sensing and solar energy production. We report a single-step immobilization process based on affinity and achieved by genetically engineering the protein of interest to carry a hematite-binding peptide. Fabricated hematite nanoparticles were then investigated for the immobilization of the two biomolecules C-phycocyanin (CPC) and laccase from Bacillus pumilus (LACC) under mild conditions. Genetic engineering of biomolecules with a hematite-affinity peptide led to a higher extent of protein immobilization and enhanced the catalytic activity of the enzyme.

  10. Reflection symmetry detection using locally affine invariant edge correspondence.

    PubMed

    Wang, Zhaozhong; Tang, Zesheng; Zhang, Xiao

    2015-04-01

    Reflection symmetry detection receives increasing attentions in recent years. The state-of-the-art algorithms mainly use the matching of intensity-based features (such as the SIFT) within a single image to find symmetry axes. This paper proposes a novel approach by establishing the correspondence of locally affine invariant edge-based features, which are superior to the intensity based in the aspects that it is insensitive to illumination variations, and applicable to textureless objects. The locally affine invariance is achieved by simple linear algebra for efficient and robust computations, making the algorithm suitable for detections under object distortions like perspective projection. Commonly used edge detectors and a voting process are, respectively, used before and after the edge description and matching steps to form a complete reflection detection pipeline. Experiments are performed using synthetic and real-world images with both multiple and single reflection symmetry axis. The test results are compared with existing algorithms to validate the proposed method.

  11. Structural origins of high-affinity biotin binding to streptavidin.

    PubMed

    Weber, P C; Ohlendorf, D H; Wendoloski, J J; Salemme, F R

    1989-01-06

    The high affinity of the noncovalent interaction between biotin and streptavidin forms the basis for many diagnostic assays that require the formation of an irreversible and specific linkage between biological macromolecules. Comparison of the refined crystal structures of apo and a streptavidin:biotin complex shows that the high affinity results from several factors. These factors include the formation of multiple hydrogen bonds and van der Waals interactions between biotin and the protein, together with the ordering of surface polypeptide loops that bury the biotin in the protein interior. Structural alterations at the biotin binding site produce quaternary changes in the streptavidin tetramer. These changes apparently propagate through cooperative deformations in the twisted beta sheets that link tetramer subunits.

  12. Robust Spectral Clustering Using Statistical Sub-Graph Affinity Model

    PubMed Central

    Eichel, Justin A.; Wong, Alexander; Fieguth, Paul; Clausi, David A.

    2013-01-01

    Spectral clustering methods have been shown to be effective for image segmentation. Unfortunately, the presence of image noise as well as textural characteristics can have a significant negative effect on the segmentation performance. To accommodate for image noise and textural characteristics, this study introduces the concept of sub-graph affinity, where each node in the primary graph is modeled as a sub-graph characterizing the neighborhood surrounding the node. The statistical sub-graph affinity matrix is then constructed based on the statistical relationships between sub-graphs of connected nodes in the primary graph, thus counteracting the uncertainty associated with the image noise and textural characteristics by utilizing more information than traditional spectral clustering methods. Experiments using both synthetic and natural images under various levels of noise contamination demonstrate that the proposed approach can achieve improved segmentation performance when compared to existing spectral clustering methods. PMID:24386111

  13. Tandem affinity purification vectors for use in gram positive bacteria.

    PubMed

    Yang, Xiao; Doherty, Geoff P; Lewis, Peter J

    2008-01-01

    Tandem affinity purification has become a valuable tool for the isolation of protein complexes. Here we describe the construction and use of a series of plasmid vectors for Gram positive bacteria. The vectors utilize the SPA tag as well as variants containing a 3C rather than the TEV protease site as 3C protease has been shown to work efficiently at the low temperatures (4 degrees C) used to isolate protein complexes. In addition, a further vector incorporates a GST moiety in place of the 3xFLAG of the SPA tag which provides an additional tagging option for situations where SPA binding may be inefficient. The vectors are all compatible with previously constructed fluorescent protein fusion vectors enabling construction of a suite of affinity and fluorescently tagged genes using a single PCR product.

  14. Complex high affinity interactions occur between MHCI and superantigens

    NASA Technical Reports Server (NTRS)

    Chapes, S. K.; Herpich, A. R.; Spooner, B. S. (Principal Investigator)

    1998-01-01

    Staphylococcal enterotoxins A and C1 (SEA or SEC1) bound to major histocompatibility-I (MHCI) molecules with high affinity (binding constants ranging from 1.1 microM to 79 nM). SEA and SEC1 directly bound MHCI molecules that had been captured by monoclonal antibodies specific for H-2Kk, H-2Dk, or both. In addition, MHCI-specific antibodies inhibited the binding of SEC1 to LM929 cells and SEA competitively inhibited SEC1 binding; indicating that the superantigens bound to MHCI on the cell surface. The affinity and number of superantigen binding sites differed depending on whether MHCI was expressed in the membrane of LM929 cells or whether it was captured. These data support the hypothesis that MHCI molecules can serve as superantigen receptors.

  15. Bioskin as an affinity matrix for the separation of glycoproteins.

    PubMed

    Vicente, C; Sebastián, B; Fontaniella, B; Márquez, A; Xavier Filho, L; Legaz, M E

    2001-05-11

    Bioskin is a natural product produced by a mixed culture of Acetobacter xylinum, Saccharomyces cerevisiae and S. pombe cultured on media containing sucrose. It is of fibrillar nature able to retain some proteins, such as cytochrome c, by adsorption, and mainly composed of glucosamine and N-acetyl-D-glucosamine. This makes it possible that, at an adequate pH value, proteins charged as polyanionic molecules, such as catalase, can be retained by ionic adsorption using the positively charged amino groups of the matrix. In addition, bioskin can also be used as an affinity matrix to retain glycoproteins able to perform specific affinity reactions with the amino sugars of the matrix, such as invertase, fetuin or ovalbumin. Its possible use as a chromatographic support is discussed.

  16. "Clickable" affinity ligands for effective separation of glycoproteins.

    PubMed

    Suksrichavalit, Thummaruk; Yoshimatsu, Keiichi; Prachayasittikul, Virapong; Bülow, Leif; Ye, Lei

    2010-06-04

    In this paper, we present a new modular approach to immobilize boronic acid ligands that can offer effective separation of glycoproteins. A new "clickable" boronic acid ligand was synthesized by introducing a terminal acetylene group into commercially available 3-aminophenyl boronic acid. The clickable ligand, 3-(prop-2-ynyloxycarbonylamino)phenylboronic acid (2) could be easily coupled to azide-functionalized hydrophilic Sepharose using Cu(I)-catalyzed 1,3-dipolar cycloaddition reaction under mild condition. Compared to other boronic acid affinity gels, the new affinity gel displayed superior effectiveness in separating model glycoproteins (ovalbumin and RNase B) from closely related bovine serum albumin and RNase A in the presence of crude Escherichia coli proteins. Because of the simplicity of the immobilization through "click chemistry", the new ligand 2 is expected to not only offer improved glycoprotein separation in other formats, but also act as a useful building block to develop new chemical sensors for analysis of other glycan compounds.

  17. Polysulfone affinity membranes for the treatment of amino acid mixtures.

    PubMed

    Rodemann, K; Staude, E

    1995-06-20

    Affinity membranes for the treatment of solutions containing amino acids were obtained via lithiating polysulfone that was subsequently converted with glycidylether. From this polymer asymmetric ultrafiltration membranes were cast. The membranes were reacted with iminodiacetic acid yielding membranes fitted out with bidentate chelates. The same reaction path was applied to commercially available symmetric microfiltration membranes. The chelate-bearing membranes were complexed with Cu, Ni, and Zn ions. For the experiments with amino acids only the Cu-complexed membranes were used. The complexation constants for histidine and tryptophan for six different membranes were determined. Because of the affinity of these two amino acids for the complexed Cu ions, they could easily be separated from solutions containing amino acids such as alanine, glycine, and valine. Also, concentrating very dilute amino acid solutions was carried out successfully.

  18. Mullerian inhibiting substance fractionation by dye affinity chromatography.

    PubMed

    Budzik, G P; Powell, S M; Kamagata, S; Donahoe, P K

    1983-08-01

    Mullerian inhibiting substance (MIS), a large glycoprotein secreted by the fetal and neonatal testis, is responsible for regression of the Mullerian ducts in the male embryo. This fetal growth regulator has been purified more than 2000-fold from crude testicular incubation medium following fractionation on a triazinyl dye affinity support. A high yield of 60% recovered activity was achieved in the absence of exogenous carrier protein by stabilizing MIS with 2-mercaptoethanol, EDTA, and Nonidet-P40 and eliminating losses in the handling and concentration of MIS fractions. Although affinity elution with nucleotides has proved successful in other systems, MIS could not be eluted with ATP, GTP, or AMP, with or without divalent metal ions. Nucleotide elution, however, does remove contaminating proteins prior to MIS recovery with high ionic strength. The 2000-fold-purified MIS fraction, although not homogeneous, shows a reduction-sensitive band after SDS-gel electrophoresis that has been proposed to be the MIS dimer.

  19. Selective high affinity polydentate ligands and methods of making such

    DOEpatents

    DeNardo, Sally; DeNardo, Gerald; Balhorn, Rodney

    2010-02-16

    This invention provides novel polydentate selective high affinity ligands (SHALs) that can be used in a variety of applications in a manner analogous to the use of antibodies. SHALs typically comprise a multiplicity of ligands that each bind different region son the target molecule. The ligands are joined directly or through a linker thereby forming a polydentate moiety that typically binds the target molecule with high selectivity and avidity.

  20. Optimizing molecular electrostatic interactions: Binding affinity and specificity

    NASA Astrophysics Data System (ADS)

    Kangas, Erik

    The design of molecules that bind tightly and specifically to designated target molecules is an important goal in many fields of molecular science. While the shape of the molecule to be designed is a relatively well defined problem with an intuitive answer, determination of the distribution of electrostatic charge that it should have in order to possess high affinity and/or specificity for a target is a subtle problem involving a tradeoff between an unfavorable electrostatic desolvation penalty incurred due to the removal of solvent from the interacting surfaces of the reactants, and the generally favorable intermolecular interactions made in the bound state. In this thesis, a theoretical formalism based on a continuum electrostatic approximation is developed in which charge distributions leading to optimal affinity and/or high specificity may be obtained. Methods for obtaining these charge distributions are developed in detail and analytical solutions are obtained in several special cases (where the molecules are shaped as infinite membranes, spheres, and spheroids). Their existence and non-uniqueness are also shown, and it is proven that the resulting optimized electrostatic binding free energies are favorable (negative) in many cases of physical interest. Affinity and specificity optimization is then applied to the chorismate mutase family of enzymes, including the catalytic antibody 1F7. It is shown that affinity optimization can be used to suggest better molecular inhibitors and that specificity optimization can be used to help elucidate molecular function and possibly aid in the creation of improved haptens. (Copies available exclusively from MIT Libraries, Rm. 14-0551, Cambridge, MA 02139-4307. Ph. 617-253-5668; Fax 617-253-1690.)

  1. Self-affinity and Crossover of A Clay Deposit

    NASA Astrophysics Data System (ADS)

    Fossum, J. O.; Huru Bergene, H.; Hansen, A.; Manificat, G.

    Self-affine dehydrated colloidal deposits on fresh mica surfaces of the synthetic 2:1 smectite clay laponite have been studied by means of Atomic Force Microscopy. AFM images of these prepared assemblies of sol and gel aggregates have been analyzed both by means of standard AFM software, and wavelet methods. The deposited surfaces show an anti-persistent to persistent crossover with a clay concentration dependent crossover length. It is thus concluded that the investigated electrolyte concentrations play a minor role

  2. Protein separation using affinity-based reversed micelles

    PubMed

    Sun; Gu; Tong; Bai; Ichikawa; Furusaki

    1999-05-01

    Reversed micellar two-phase extraction is a developing technique for protein separation. Introduction of an affinity ligand is considered to be an effective approach to increase the selectivity and capacity of reversed micelles. In this article, Cibacron Blue F3G-A (CB) as an affinity ligand was immobilized to reversed micelles composed of soybean lecithin by a two-phase reaction. The affinity partitioning of lysozyme and bovine serum albumin (BSA) to the CB-lecithin micelles was studied. Formation of mixed micelles by additionally introducing a nonionic surfactant, Tween 85, to the CB-lecithin micelles was effective to increase the solubilization of lysozyme due to the increase of W0 (water/surfactant molar ratio)/micellar size. The partitioning isotherms of lysozyme to the CB-lecithin micelles with and without Tween 85 were expressed by the Langmuir equation. The dissociation constants in the Langmuir equation decreased on addition of Tween 85, indicating the increase of the effectiveness of lysozyme binding to the immobilized CB. On addition of 20 g/L Tween 85 to 50 g/L lecithin/hexane micellar phase containing 0.1 mmol/L CB, the extraction capacity for lysozyme could be increased by 42%. Moreover, the CB-lecithin micelles with or without Tween 85 showed significant size exclusion for BSA due to its high molecular weight. Thus, lysozyme and BSA were separated from artificial solutions containing the two proteins. In addition, the affinity-based reversed micellar phase containing Tween 85 was recycled three times for lysozyme purification from crude egg-white solutions. Lysozyme purity increased by 16-18-fold, reaching 60-70% in the recycled use.

  3. Glycopeptide Site Heterogeneity and Structural Diversity Determined by Combined Lectin Affinity Chromatography/IMS/CID/MS Techniques

    NASA Astrophysics Data System (ADS)

    Zhu, Feifei; Trinidad, Jonathan C.; Clemmer, David E.

    2015-07-01

    Glycopeptides from a tryptic digest of chicken ovomucoid were enriched using a simplified lectin affinity chromatography (LAC) platform, and characterized by high-resolution mass spectrometry (MS) as well as ion mobility spectrometry (IMS)-MS. The LAC platform effectively enriched the glycoproteome, from which a total of 117 glycopeptides containing 27 glycan forms were identified for this protein. IMS-MS analysis revealed a high degree of glycopeptide site heterogeneity. Comparison of the IMS distributions of the glycopeptides from different charge states reveals that higher charge states allow more structures to be resolved. Presumably the repulsive interactions between charged sites lead to more open configurations, which are more readily separated compared with the more compact, lower charge state forms of the same groups of species. Combining IMS with collision induced dissociation (CID) made it possible to determine the presence of isomeric glycans and to reconstruct their IMS profiles. This study illustrates a workflow involving hybrid techniques for determining glycopeptide site heterogeneity and evaluating structural diversity of glycans and glycopeptides.

  4. Methylated-antibody affinity purification to improve proteomic identification of plant RNA polymerase Pol V complex and the interacting proteins

    PubMed Central

    Qin, Guochen; Ma, Jun; Chen, Xiaomei; Chu, Zhaoqing; She, Yi-Min

    2017-01-01

    Affinity purification followed by enzymatic digestion and mass spectrometry has been widely utilized for the sensitive detection of interacting proteins and protein complexes in various organisms. In plants, the method is technically challenging due to the low abundance proteins, non-specific binding and difficulties of eluting interacting proteins from antibody beads. In this report, we describe a strategy to modify antibodies by reductive methylation of lysines without affecting their binding properties, followed by on-bead digestion of bound proteins with endoproteinase Lys-C. By this method, the antibody remains intact and does not interfere with the downstream identification of interacting proteins. Non-specific binding proteins were excluded using 14N/15N-metabolic labeling of wild-type and the transgenic plant counterparts. The method was employed to identify 12 co-immunoprecipitated protein subunits in Pol V complex and to discover 17 potential interacting protein targets in Arabidopsis. Our results demonstrated that the modification of antibodies by reductive dimethylation can improve the reliability and sensitivity of identifying low-abundance proteins through on-bead digestion and mass spectrometry. We also show that coupling this technique with chemical crosslinking enables in-depth characterization of endogenous protein complexes and the protein-protein interaction networks including mapping the surface topology and post-translational modifications of interacting proteins. PMID:28224978

  5. Assessing high affinity binding to HLA-DQ2.5 by a novel peptide library based approach.

    PubMed

    Jüse, Ulrike; Arntzen, Magnus; Højrup, Peter; Fleckenstein, Burkhard; Sollid, Ludvig M

    2011-04-01

    Here we report on a novel peptide library based method for HLA class II binding motif identification. The approach is based on water soluble HLA class II molecules and soluble dedicated peptide libraries. A high number of different synthetic peptides are competing to interact with a limited amount of HLA molecules, giving a selective force in the binding. The peptide libraries can be designed so that the sequence length, the alignment of binding registers, the numbers and composition of random positions are controlled, and also modified amino acids can be included. Selected library peptides bound to HLA are then isolated by size exclusion chromatography and sequenced by tandem mass spectrometry online coupled to liquid chromatography. The MS/MS data are subsequently searched against a library defined database using a search engine such as Mascot, followed by manual inspection of the results. We used two dodecamer and two decamer peptide libraries and HLA-DQ2.5 to test possibilities and limits of this method. The selected sequences which we identified in the fraction eluted from HLA-DQ2.5 showed a higher average of their predicted binding affinity values compared to the original peptide library. The eluted sequences fit very well with the previously described HLA-DQ2.5 peptide binding motif. This novel method, limited by library complexity and sensitivity of mass spectrometry, allows the analysis of several thousand synthetic sequences concomitantly in a simple water soluble format.

  6. Methylated-antibody affinity purification to improve proteomic identification of plant RNA polymerase Pol V complex and the interacting proteins.

    PubMed

    Qin, Guochen; Ma, Jun; Chen, Xiaomei; Chu, Zhaoqing; She, Yi-Min

    2017-02-22

    Affinity purification followed by enzymatic digestion and mass spectrometry has been widely utilized for the sensitive detection of interacting proteins and protein complexes in various organisms. In plants, the method is technically challenging due to the low abundance proteins, non-specific binding and difficulties of eluting interacting proteins from antibody beads. In this report, we describe a strategy to modify antibodies by reductive methylation of lysines without affecting their binding properties, followed by on-bead digestion of bound proteins with endoproteinase Lys-C. By this method, the antibody remains intact and does not interfere with the downstream identification of interacting proteins. Non-specific binding proteins were excluded using (14)N/(15)N-metabolic labeling of wild-type and the transgenic plant counterparts. The method was employed to identify 12 co-immunoprecipitated protein subunits in Pol V complex and to discover 17 potential interacting protein targets in Arabidopsis. Our results demonstrated that the modification of antibodies by reductive dimethylation can improve the reliability and sensitivity of identifying low-abundance proteins through on-bead digestion and mass spectrometry. We also show that coupling this technique with chemical crosslinking enables in-depth characterization of endogenous protein complexes and the protein-protein interaction networks including mapping the surface topology and post-translational modifications of interacting proteins.

  7. Recombinant spider silk genetically functionalized with affinity domains.

    PubMed

    Jansson, Ronnie; Thatikonda, Naresh; Lindberg, Diana; Rising, Anna; Johansson, Jan; Nygren, Per-Åke; Hedhammar, My

    2014-05-12

    Functionalization of biocompatible materials for presentation of active protein domains is an area of growing interest. Herein, we describe a strategy for functionalization of recombinant spider silk via gene fusion to affinity domains of broad biotechnological use. Four affinity domains of different origin and structure; the IgG-binding domains Z and C2, the albumin-binding domain ABD, and the biotin-binding domain M4, were all successfully produced as soluble silk fusion proteins under nondenaturing purification conditions. Silk films and fibers produced from the fusion proteins were demonstrated to be chemically and thermally stable. Still, the bioactive domains are concluded to be folded and accessible, since their respective targets could be selectively captured from complex samples, including rabbit serum and human plasma. Interestingly, materials produced from mixtures of two different silk fusion proteins displayed combined binding properties, suggesting that tailor-made materials with desired stoichiometry and surface distributions of several binding domains can be produced. Further, use of the IgG binding ability as a general mean for presentation of desired biomolecules could be demonstrated for a human vascular endothelial growth factor (hVEGF) model system, via a first capture of anti-VEGF IgG to silk containing the Z-domain, followed by incubation with hVEGF. Taken together, this study demonstrates the potential of recombinant silk, genetically functionalized with affinity domains, for construction of biomaterials capable of presentation of almost any desired biomolecule.

  8. Measurement of the Electron Affinities of Indium and Thallium

    SciTech Connect

    Thompson, J. S.; Carpenter, D. L.; Covington, A. M.; Williams, W. W.; Kvale, T. J.; Seely, D. G.

    1999-03-20

    The electron affinities of indium and thallium were measured in separate experiments using the laser-photodetachment electron spectroscopy technique. The measurements were performed at the University of Nevada, Reno. Negative ion beams of both indium and thallium were extracted from a cesium-sputter negative ion source, and mass analyzed using a 90{sup o} bending magnet. The negative ion beam of interest was then crossed at 90{sup o} with a photon beam from a cw 25-Watt Ar{sup +} laser. The resulting photoelectrons were energy analyzed with a 160{sup o} spherical-sector spectrometer. The electron affinity of In({sup 2}P{sub 1/2}) was determined to be 0.404 {+-} 0.009 eV and the electron affinity of thallium was determined to be 0.377 {+-} 0.013 eV. The fine-structure splittings in the ground states of the negative ions were also determined. The experimental measurements will be compared to several recent theoretical predictions.

  9. Affine sphere spacetimes which satisfy the relativity principle

    NASA Astrophysics Data System (ADS)

    Minguzzi, E.

    2017-01-01

    In the context of Lorentz-Finsler spacetime theories the relativity principle holds at a spacetime point if the indicatrix (observer space) is homogeneous. We point out that in four spacetime dimensions there are just three kinematical models which respect an exact form of the relativity principle and for which all observers agree on the spacetime volume. They have necessarily affine sphere indicatrices. For them every observer which looks at a flash of light emitted by a point would observe, respectively, an expanding (a) sphere, (b) tetrahedron, or (c) cone, with barycenter at the point. The first model corresponds to Lorentzian relativity, the second one has been studied by several authors though the relationship with affine spheres passed unnoticed, and the last one has not been previously recognized and it is studied here in some detail. The symmetry groups are O+(3 ,1 ),R3,O+(2 ,1 )×R , respectively. In the second part, devoted to the general relativistic theory, we show that the field equations can be obtained by gauging the Finsler Lagrangian symmetry while avoiding direct use of Finslerian curvatures. We construct some notable affine sphere spacetimes which in the appropriate velocity limit return the Schwarzschild, Kerr-Schild, Kerr-de Sitter, Kerr-Newman, Taub, and Friedmann-Lemaître-Robertson-Walker spacetimes, respectively.

  10. Affinity-tuning leukocyte integrin for development of safe therapeutics

    NASA Astrophysics Data System (ADS)

    Park, Spencer

    Much attention has been given to the molecular and cellular pathways linking inflammation with cancer and the local tumor environment to identify new target molecules that could lead to improved diagnosis and treatment. Among the many molecular players involved in the complex response, central to the induction of inflammation is intercellular adhesion molecule (ICAM)-1, which is of particular interest for its highly sensitive and localized expression in response to inflammatory signals. ICAM-1, which has been implicated to play a critical role in tumor progression in various types of cancer, has also been linked to cancer metastases, where ICAM-1 facilitates the spread of metastatic cancer cells to secondary sites. This unique expression profile of ICAM-1 throughout solid tumor microenvironment makes ICAM-1 an intriguing molecular target, which holds great potential as an important diagnostic and therapeutic tool. Herein, we have engineered the ligand binding domain, or the inserted (I) domain of a leukocyte integrin, to exhibit a wide range of monovalent affinities to the natural ligand, ICAM-1. Using the resulting I domain variants, we have created drug and gene delivery nanoparticles, as well as targeted immunotherapeutics that have the ability to bind and migrate to inflammatory sites prevalent in tumors and the associated microenvironment. Through the delivery of diagnostic agents, chemotherapeutics, and immunotherapeutics, the following chapters demonstrate that the affinity enhancements achieved by directed evolution bring the affinity of I domains into the range optimal for numerous applications.

  11. Generation of recombinant antibodies and means for increasing their affinity.

    PubMed

    Altshuler, E P; Serebryanaya, D V; Katrukha, A G

    2010-12-01

    Highly specific interaction with foreign molecules is a unique feature of antibodies. Since 1975, when Keller and Milstein proposed the method of hybridoma technology and prepared mouse monoclonal antibodies, many antibodies specific to various antigens have been obtained. Recent development of methods for preparation of recombinant DNA libraries and in silico bioinformatics approaches for protein structure analysis makes possible antibody preparation using gene engineering approaches. The development of gene engineering methods allowed creating recombinant antibodies and improving characteristics of existing antibodies; this significantly extends the applicability of antibodies. By modifying biochemical and immunochemical properties of antibodies by changing their amino acid sequences it is possible to create antibodies with properties optimal for certain tasks. For example, application of recombinant technologies resulted in antibody preparation of high affinity significantly exceeding the initial affinity of natural antibodies. In this review we summarize information about the structure, modes of preparation, and application of recombinant antibodies and their fragments and also consider the main approaches used to increase antibody affinity.

  12. Structure of Greyhound hemoglobin: origin of high oxygen affinity.

    PubMed

    Bhatt, Veer S; Zaldívar-López, Sara; Harris, David R; Couto, C Guillermo; Wang, Peng G; Palmer, Andre F

    2011-05-01

    This study presents the crystal structure of Greyhound hemoglobin (GrHb) determined to 1.9 Å resolution. GrHb was found to crystallize with an α₁β₁ dimer in the asymmetric unit and belongs to the R2 state. Oxygen-affinity measurements combined with the fact that GrHb crystallizes in the R2 state despite the high-salt conditions used for crystallization strongly indicate that GrHb can serve as a model high-oxygen-affinity hemoglobin (Hb) for higher mammals, especially humans. Structural analysis of GrHb and its comparison with the R2-state of human Hb revealed several regions that can potentially contribute to the high oxygen affinity of GrHb and serve to rationalize the additional stability of the R2-state of GrHb. A previously well studied hydrophobic cluster of bar-headed goose Hb near α119 was also incorporated in the comparison between GrHb and human Hb. Finally, a structural comparison with generic dog Hb and maned wolf Hb was conducted, revealing that in contrast to GrHb these structures belong to the R state of Hb and raising the intriguing possibility of an additional allosteric factor co-purifying with GrHb that can modulate its quaternary structure.

  13. High-throughput fragment screening by affinity LC-MS.

    PubMed

    Duong-Thi, Minh-Dao; Bergström, Maria; Fex, Tomas; Isaksson, Roland; Ohlson, Sten

    2013-02-01

    Fragment screening, an emerging approach for hit finding in drug discovery, has recently been proven effective by its first approved drug, vemurafenib, for cancer treatment. Techniques such as nuclear magnetic resonance, surface plasmon resonance, and isothemal titration calorimetry, with their own pros and cons, have been employed for screening fragment libraries. As an alternative approach, screening based on high-performance liquid chromatography separation has been developed. In this work, we present weak affinity LC/MS as a method to screen fragments under high-throughput conditions. Affinity-based capillary columns with immobilized thrombin were used to screen a collection of 590 compounds from a fragment library. The collection was divided into 11 mixtures (each containing 35 to 65 fragments) and screened by MS detection. The primary screening was performed in <4 h (corresponding to >3500 fragments per day). Thirty hits were defined, which subsequently entered a secondary screening using an active site-blocked thrombin column for confirmation of specificity. One hit showed selective binding to thrombin with an estimated dissociation constant (K (D)) in the 0.1 mM range. This study shows that affinity LC/MS is characterized by high throughput, ease of operation, and low consumption of target and fragments, and therefore it promises to be a valuable method for fragment screening.

  14. Composite affinity sorbents and their cleaning in place.

    PubMed

    Girot, P; Moroux, Y; Duteil, X P; Nguyen, C; Boschetti, E

    1990-06-27

    Making large-scale affinity sorbents that are reusable under acceptable hygienic conditions implies specific treatments for cleaning in place with known aqueous solutions of chemical agents. However, common agents such as sodium hydroxide are frequently considered too drastic for the stability of macromolecular biologically active immobilized ligands. According to a large series of trials, it was found that only a mixture of sodium hydroxide and ethanol was actually effective in sterilizing a sorbent in a single step. When hydroxide or an ethanol-acetic acid mixture were used alone, they were not totally efficient in the inactivation of sporulated Bacillus subtilis. Conversely, they were efficient when used sequentially. All these solutions were able to remove pyrogens from chromatographic sorbents. As the sterilizing solutions contained a certain amount of ethanol, the most suitable chromatographic affinity sorbents had to be based on an incompressible matrix. When washing an affinity silica sorbent that had proteins as ligands with solutions such as sodium hydroxide, ethanol-acetic acid or ethanol-sodium hydroxide, it was found that certain sorbents were able to tolerate the treatments without a noticeable decrease in their biochemical activity.

  15. Conformational equilibria and intrinsic affinities define integrin activation.

    PubMed

    Li, Jing; Su, Yang; Xia, Wei; Qin, Yan; Humphries, Martin J; Vestweber, Dietmar; Cabañas, Carlos; Lu, Chafen; Springer, Timothy A

    2017-03-01

    We show that the three conformational states of integrin α5β1 have discrete free energies and define activation by measuring intrinsic affinities for ligand of each state and the equilibria linking them. The 5,000-fold higher affinity of the extended-open state than the bent-closed and extended-closed states demonstrates profound regulation of affinity. Free energy requirements for activation are defined with protein fragments and intact α5β1 On the surface of K562 cells, α5β1 is 99.8% bent-closed. Stabilization of the bent conformation by integrin transmembrane and cytoplasmic domains must be overcome by cellular energy input to stabilize extension. Following extension, headpiece opening is energetically favored. N-glycans and leg domains in each subunit that connect the ligand-binding head to the membrane repel or crowd one another and regulate conformational equilibria in favor of headpiece opening. The results suggest new principles for regulating signaling in the large class of receptors built from extracellular domains in tandem with single-span transmembrane domains.

  16. Pepsin-modified chiral monolithic column for affinity capillary electrochromatography.

    PubMed

    Hong, Tingting; Chi, Cuijie; Ji, Yibing

    2014-11-01

    Pepsin-modified affinity monolithic capillary electrochromatography, a novel microanalysis system, was developed by the covalent bonding of pepsin on silica monolith. The column was successfully applied in the chiral separation of (±)-nefopam. Furthermore, the electrochromatographic performance of the pepsin-functionalized monolith for enantiomeric analysis was evaluated in terms of protein content, pH of running buffer, sample volume, buffer concentration, applied voltage, and capillary temperature. The relative standard deviation (%RSD) values of retention time (intraday <0.53, n = 10; interday <0.53, n = 10; column-to-column <0.70, n = 20; and batch-to-batch <0.80, n = 20) indicated satisfactory stability of these columns. No appreciable change was observed in retention and resolution for chiral recognition of (±)-nefopam in 50 days with 100 injections. The proteolytic activity of this stationary phase was further characterized with bovine serum albumin as substrate for online protein digestion. As for monolithic immobilized enzyme reactor, successive protein injections confirmed both the operational stability and ability to reuse the bioreactor for at least 20 digestions. It implied that the affinity monolith used in this research opens a new path of exploring particularly versatile class of enzymes to develop enzyme-modified affinity capillary monolith for enantioseparation.

  17. Relative binding affinities of monolignols to horseradish peroxidase

    SciTech Connect

    Sangha, Amandeep K.; Petridis, Loukas; Cheng, Xiaolin; Smith, Jeremy C.

    2016-07-22

    Monolignol binding to the peroxidase active site is the first step in lignin polymerization in plant cell walls. Using molecular dynamics, docking, and free energy perturbation calculations, we investigate the binding of monolignols to horseradish peroxidase C. Our results suggest that p-coumaryl alcohol has the strongest binding affinity followed by sinapyl and coniferyl alcohol. Stacking interactions between the monolignol aromatic rings and nearby phenylalanine residues play an important role in determining the calculated relative binding affinities. p-Coumaryl and coniferyl alcohols bind in a pose productive for reaction in which a direct H-bond is formed between the phenolic –OH group and a water molecule (W2) that may facilitate proton transfer during oxidation. In contrast, in the case of sinapyl alcohol there is no such direct interaction, the phenolic –OH group instead interacting with Pro139. Furthermore, since proton and electron transfer is the rate-limiting step in monolignol oxidation by peroxidase, the binding pose (and thus the formation of near attack conformation) appears to play a more important role than the overall binding affinity in determining the oxidation rate.

  18. Relative binding affinities of monolignols to horseradish peroxidase

    DOE PAGES

    Sangha, Amandeep K.; Petridis, Loukas; Cheng, Xiaolin; ...

    2016-07-22

    Monolignol binding to the peroxidase active site is the first step in lignin polymerization in plant cell walls. Using molecular dynamics, docking, and free energy perturbation calculations, we investigate the binding of monolignols to horseradish peroxidase C. Our results suggest that p-coumaryl alcohol has the strongest binding affinity followed by sinapyl and coniferyl alcohol. Stacking interactions between the monolignol aromatic rings and nearby phenylalanine residues play an important role in determining the calculated relative binding affinities. p-Coumaryl and coniferyl alcohols bind in a pose productive for reaction in which a direct H-bond is formed between the phenolic –OH group andmore » a water molecule (W2) that may facilitate proton transfer during oxidation. In contrast, in the case of sinapyl alcohol there is no such direct interaction, the phenolic –OH group instead interacting with Pro139. Furthermore, since proton and electron transfer is the rate-limiting step in monolignol oxidation by peroxidase, the binding pose (and thus the formation of near attack conformation) appears to play a more important role than the overall binding affinity in determining the oxidation rate.« less

  19. Affinity approaches in RNAi-based therapeutics purification.

    PubMed

    Pereira, Patrícia; Queiroz, João A; Figueiras, Ana; Sousa, Fani

    2016-05-15

    The recent investigation on RNA interference (RNAi) related mechanisms and applications led to an increased awareness of the importance of RNA in biology. Nowadays, RNAi-based technology has emerged as a potentially powerful tool for silencing gene expression, being exploited to develop new therapeutics for treating a vast number of human disease conditions, as it is expected that this technology can be translated onto clinical applications in a near future. This approach makes use of a large number of small (namely short interfering RNAs, microRNAs and PIWI-interacting RNAs) and long non-coding RNAs (ncRNAs), which are likely to have a crucial role as the next generation therapeutics. The commercial and biomedical interest in these RNAi-based therapy applications have fostered the need to develop innovative procedures to easily and efficiently purify RNA, aiming to obtain the final product with high purity degree, good quality and biological activity. Recently, affinity chromatography has been applied to ncRNAs purification, in view of the high specificity. Therefore, this article intends to review the biogenesis pathways of regulatory ncRNAs and also to discuss the most significant and recent developments as well as applications of affinity chromatography in the challenging task of purifying ncRNAs. In addition, the importance of affinity chromatography in ncRNAs purification is addressed and prospects for what is forthcoming are presented.

  20. Beyond hairballs: the use of quantitative mass spectrometry data to understand protein-protein interactions

    PubMed Central

    Gingras, Anne-Claude; Raught, Brian

    2012-01-01

    The past 10 years have witnessed a dramatic proliferation in the availability of protein interaction data. However, for interaction mapping based on affinity purification coupled with mass spectrometry (AP-MS), there is a wealth of information present in the datasets that often goes unrecorded in public repositories, and as such remains largely unexplored. Further, how this type of data is represented and used by bioinformaticians has not been well established. Here, we point out some common mistakes in how AP-MS data are handled, and describe how protein complex organization and interaction dynamics can be inferred using quantitative AP-MS approaches. PMID:22710165

  1. Genetically encoded protein photocrosslinker with a transferable mass spectrometry-identifiable label

    PubMed Central

    Yang, Yi; Song, Haiping; He, Dan; Zhang, Shuai; Dai, Shizhong; Lin, Shixian; Meng, Rong; Wang, Chu; Chen, Peng R.

    2016-01-01

    Coupling photocrosslinking reagents with mass spectrometry has become a powerful tool for studying protein–protein interactions in living systems, but it still suffers from high rates of false-positive identifications as well as the lack of information on interaction interface due to the challenges in deciphering crosslinking peptides. Here we develop a genetically encoded photo-affinity unnatural amino acid that introduces a mass spectrometry-identifiable label (MS-label) to the captured prey proteins after photocrosslinking and prey–bait separation. This strategy, termed IMAPP (In-situ cleavage and MS-label transfer After Protein Photocrosslinking), enables direct identification of photo-captured substrate peptides that are difficult to uncover by conventional genetically encoded photocrosslinkers. Taking advantage of the MS-label, the IMAPP strategy significantly enhances the confidence for identifying protein–protein interactions and enables simultaneous mapping of the binding interface under living conditions. PMID:27460181

  2. Robust adaptive control for a class of uncertain non-affine nonlinear systems using affine-type neural networks

    NASA Astrophysics Data System (ADS)

    Zhao, Shitie; Gao, Xianwen

    2016-08-01

    A robust adaptive control is proposed for a class of single-input single-output non-affine nonlinear systems. In order to approximate the unknown nonlinear function, a novel affine-type neural network is used, and then to compensate the approximation error and external disturbance a robust control term is employed. By Lyapunov stability analysis for the closed-loop system, it is proved that tracking errors asymptotically converge to zero. Moreover, an observer is designed to estimate the system states because all the states may not be available for measurements. Furthermore, the adaptation laws of neural networks and the robust controller are given out based on the Lyapunov stability theory. Finally, two simulation examples are presented to demonstrate the effectiveness of the proposed control method.

  3. 77 FR 28411 - Adrenalina, Affinity Technology Group, Inc., Braintech, Inc., Builders Transport, Incorporated...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-05-14

    ... From the Federal Register Online via the Government Publishing Office SECURITIES AND EXCHANGE COMMISSION Adrenalina, Affinity Technology Group, Inc., Braintech, Inc., Builders Transport, Incorporated... concerning the securities of Affinity Technology Group, Inc. because it has not filed any periodic...

  4. The Experimental Values of Atomic Electron Affinities: Their Selection and Periodic Behavior

    ERIC Educational Resources Information Center

    Chen, E. C. M.; Wentworth, W. E.

    1975-01-01

    Describes new experimental techniques, such as dye-laser photodetachment studies, which have recently provided accurate values for the electron affinity of numerous elements. Provides a periodic chart with available electron affinity data. (MLH)

  5. The Purification of a Blood Group A Glycoprotein: An Affinity Chromatography Experiment.

    ERIC Educational Resources Information Center

    Estelrich, J.; Pouplana, R.

    1988-01-01

    Describes a purification process through affinity chromatography necessary to obtain specific blood group glycoproteins from erythrocytic membranes. Discusses the preparation of erythrocytic membranes, extraction of glycoprotein from membranes, affinity chromatography purification, determination of glycoproteins, and results. (CW)

  6. Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation.

    PubMed

    Zielonka, Stefan; Weber, Niklas; Becker, Stefan; Doerner, Achim; Christmann, Andreas; Christmann, Christine; Uth, Christina; Fritz, Janine; Schäfer, Elena; Steinmann, Björn; Empting, Martin; Ockelmann, Pia; Lierz, Michael; Kolmar, Harald

    2014-12-10

    A novel method for stepwise in vitro affinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterized via thermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generated in vitro without largely compromising the desirable high thermostability of the vNAR scaffold.

  7. Monitoring cleavage of fusion proteins by matrix-assisted laser desorption ionization/mass spectrometry: recombinant HIV-1IIIB p26.

    PubMed

    Parker, C E; Papac, D I; Tomer, K B

    1996-07-15

    Matrix-associated laser desorption ionization/mass spectrometry (MALDI/MS) has been used to examine whole bacteria for the presence of a recombinant HIV p26 fusion protein. MALDI/MS, combined with affinity-purification techniques, is also shown to be very useful in monitoring the enzymatic cleavage of both affinity-bound fusion protein and fusion protein in solution. The combination of mass resolution, sensitivity, and speed of analysis makes MALDI/MS an attractive alternative to SDS-PAGE.

  8. Mass spectrometry and tandem mass spectrometry of citrus limonoids.

    PubMed

    Tian, Qingguo; Schwartz, Steven J

    2003-10-15

    Methods for atmospheric pressure chemical ionization tandem mass spectrometry (APCI-MS/MS) of citrus limonoid aglycones and electrospray ionization tandem mass spectrometry (ESI-MS/MS) of limonoid glucosides are reported. The fragmentation patterns of four citrus limonoid aglycones (limonin, nomilin, obacunone, and deacetylnomilin) and six limonoid glucosides, that is, limonin 17-beta-D-glucopyranoside (LG), nomilin 17-beta-D-glucopyranoside (NG), nomilinic acid 17-beta-D-glucopyranoside (NAG), deacetyl nomilinic acid 17-beta-D-glucopyranoside (DNAG), obacunone 17-beta-D-glucopyranoside (OG), and obacunoic acid 17-beta-D-glucopyranoside (OAG) were investigated using a quadruple mass spectrometer in low-energy collisionally activated dissociation (CAD). The four limonoid aglycones and four limonoid glucosides (LG, OG, NAG, and DNAG) were purified from citrus seeds; the other two limonoid glucosides (NG and OAG) were tentatively identified in the crude extract of grapefruit seeds by ESI mass spectrometry in both positive and negative ion analysis. Ammonium hydroxide or acetic acid was added to the mobile phase to facilitate ionization. During positive ion APCI analysis of limonoid aglycones, protonated molecular ion, [M + H]+, or adduct ion, [M + NH3 + H]-, was formed as base peaks when ammonium hydroxide was added to the mobile phase. Molecular anions or adduct ions with acetic acid ([M + HOAc - H] and [M + HOAc]-) or a deprotonated molecular ion were produced during negative ion APCI analysis of limonoid aglycones, depending on the mobile-phase modifier used. Positive ion ESI-MS of limonoid glucosides produced adduct ions of [M + H + NH3]+, [M + Na]+, and [M + K]+ when ammonium hydroxide was added to the mobile phase. After collisionally activated dissociation (CAD) of the limonoid aglycone molecular ions in negative ion APCI analysis, fragment ions indicated structural information of the precursor ions, showing the presence of methyl, carboxyl, and oxygenated ring

  9. Rapid characterization of protein chips using microwave-assisted protein tryptic digestion and MALDI mass spectrometry.

    PubMed

    Ha, Na Young; Kim, Shin Hye; Lee, Tae Geol; Han, Sang Yun

    2011-08-16

    We demonstrate that the microwave-assisted protein enzymatic digestion (MAPED) method can be successfully applied to the mass spectrometric characterization of proteins captured on the affinity surfaces of protein chips. The microwave-assisted on-chip tryptic digestion method was developed using a domestic microwave, completing the on-chip proteolysis reaction in minutes, whereas the previous on-chip digestion methods by incubation took hours of incubation time. For the model protein chips, antibody-presenting surfaces were prepared, where anti-α-tubulin1 and antibovine serum albumin (BSA) were immobilized on self-assembled monolayers. The resulting digestion efficiency, displaying sequence coverages of 30 and 14% for α-tubulin1 and BSA, respectively, was comparable to the previous time-consuming incubation studies. It allowed the characterization of immunosensed proteins by MASCOT search using peptide mass fingerprinting. In an example of this method for protein chip applications, BSA naturally involved in fetal bovine serum was unambiguously identified on a model protein chip by imaging mass spectrometry. This work shows that biomass spectrometry techniques can be implemented for surface mass spectrometry and biochip applications. Along with recent advances in imaging mass spectrometry, this technique will provide a new opportunity for high-speed, and thus high-throughput in the future, label-free mass spectrometric assays using protein arrays.

  10. Sensitive Targeted Quantification of ERK Phosphorylation Dynamics and Stoichiometry in Human Cells without Affinity Enrichment

    SciTech Connect

    Shi, Tujin; Gao, Yuqian; Gaffrey, Matthew J.; Nicora, Carrie D.; Fillmore, Thomas L.; Chrisler, William B.; Gritsenko, Marina A.; Wu, Chaochao; He, Jintang; Bloodsworth, Kent J.; Zhao, Rui; Camp II, David G.; Liu, Tao; Rodland, Karin D.; Smith, Richard D.; Wiley, H. Steven; Qian, Weijun

    2014-12-17

    Mass spectrometry-based targeted quantification is a promising technology for site-specific quantification of posttranslational modifications (PTMs). However, a major constraint of most targeted MS approaches is the limited sensitivity for quantifying low-abundance PTMs, requiring the use of affinity reagents to enrich specific PTMs. Herein, we demonstrate the direct site-specific quantification of ERK phosphorylation isoforms (pT, pY, pTpY) and their relative stoichiometries using a highly sensitive targeted MS approach termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM). PRISM provides effective enrichment of target peptides within a given fraction from complex biological matrix with minimal sample losses, followed by selected reaction monitoring (SRM) quantification. The PRISM-SRM approach enabled direct quantification of ERK phosphorylation in human mammary epithelial cells (HMEC) from as little as 25 µg tryptic peptides from whole cell lysates. Compared to immobilized metal-ion affinity chromatography, PRISM provided >10-fold improvement in signal intensities, presumably due to the better peptide recovery of PRISM for handling small size samples. This approach was applied to quantify ERK phosphorylation dynamics in HMEC treated by different doses of EGF at both the peak activation (10 min) and steady state (2 h). At 10 min, the maximal ERK activation was observed with 0.3 ng/mL dose, whereas the maximal steady state level of ERK activation at 2 h was at 3 ng/ml dose, corresponding to 1200 and 9000 occupied receptors, respectively. At 10 min, the maximally activated pTpY isoform represented ~40% of total ERK, falling to less than 10% at 2 h. The time course and dose-response profiles of individual phosphorylated ERK isoforms indicated that singly phosphorylated pT-ERK never increases significantly, while the increase of pY-ERK paralleled that of pTpY-ERK. This data supports for a processive, rather than

  11. Sensitive Targeted Quantification of ERK Phosphorylation Dynamics and Stoichiometry in Human Cells without Affinity Enrichment

    DOE PAGES

    Shi, Tujin; Gao, Yuqian; Gaffrey, Matthew J.; ...

    2014-12-17

    Mass spectrometry-based targeted quantification is a promising technology for site-specific quantification of posttranslational modifications (PTMs). However, a major constraint of most targeted MS approaches is the limited sensitivity for quantifying low-abundance PTMs, requiring the use of affinity reagents to enrich specific PTMs. Herein, we demonstrate the direct site-specific quantification of ERK phosphorylation isoforms (pT, pY, pTpY) and their relative stoichiometries using a highly sensitive targeted MS approach termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM). PRISM provides effective enrichment of target peptides within a given fraction from complex biological matrix with minimal sample losses, followed by selected reactionmore » monitoring (SRM) quantification. The PRISM-SRM approach enabled direct quantification of ERK phosphorylation in human mammary epithelial cells (HMEC) from as little as 25 µg tryptic peptides from whole cell lysates. Compared to immobilized metal-ion affinity chromatography, PRISM provided >10-fold improvement in signal intensities, presumably due to the better peptide recovery of PRISM for handling small size samples. This approach was applied to quantify ERK phosphorylation dynamics in HMEC treated by different doses of EGF at both the peak activation (10 min) and steady state (2 h). At 10 min, the maximal ERK activation was observed with 0.3 ng/mL dose, whereas the maximal steady state level of ERK activation at 2 h was at 3 ng/ml dose, corresponding to 1200 and 9000 occupied receptors, respectively. At 10 min, the maximally activated pTpY isoform represented ~40% of total ERK, falling to less than 10% at 2 h. The time course and dose-response profiles of individual phosphorylated ERK isoforms indicated that singly phosphorylated pT-ERK never increases significantly, while the increase of pY-ERK paralleled that of pTpY-ERK. This data supports for a processive, rather than

  12. PHARMACEUTICAL AND BIOMEDICAL APPLICATIONS OF AFFINITY CHROMATOGRAPHY: RECENT TRENDS AND DEVELOPMENTS

    PubMed Central

    Hage, David S.; Anguizola, Jeanethe A.; Bi, Cong; Li, Rong; Matsuda, Ryan; Papastavros, Efthimia; Pfaunmiller, Erika; Vargas, John; Zheng, Xiwei

    2012-01-01

    Affinity chromatography is a separation technique that has become increasingly important in work with biological samples and pharmaceutical agents. This method is based on the use of a biologically-related agent as a stationary phase to selectively retain analytes or to study biological interactions. This review discusses the basic principles behind affinity chromatography and examines recent developments that have occurred in the use of this method for biomedical and pharmaceutical analysis. Techniques based on traditional affinity supports are discussed, but an emphasis is placed on methods in which affinity columns are used as part of HPLC systems or in combination with other analytical methods. General formats for affinity chromatography that are considered include step elution schemes, weak affinity chromatography, affinity extraction and affinity depletion. Specific separation techniques that are examined include lectin affinity chromatography, boronate affinity chromatography, immunoaffinity chromatography, and immobilized metal ion affinity chromatography. Approaches for the study of biological interactions by affinity chromatography are also presented, such as the measurement of equilibrium constants, rate constants, or competition and displacement effects. In addition, related developments in the use of immobilized enzyme reactors, molecularly imprinted polymers, dye ligands and aptamers are briefly considered. PMID:22305083

  13. EMERGING POLLUTANTS, MASS SPECTROMETRY, AND ...

    EPA Pesticide Factsheets

    Historically fundamental to amassing our understanding of environmental processes and chemical pollution is the realm of mass spectrometry (MS) - the mainstay of analytical chemistry - the workhorse that supplies definitive data that environmental scientists and engineers reply upon for identifying molecular compositions (and ultimately structures) of chemicals. While the power of MS has long been visible to the practicing environmental chemist, it borders on obscurity to the lay public and many scientists. While MS has played a long, historic (and largely invisible) role in establishing our knowledge of environmental processes and pollution, what recognition it does enjoy is usually relegated to that of a tool. It is usually the relevance or significance of the knowledge acquired from the application of the tool that has ultimate meaning to the public and science at large - not how the data were acquired. Methods (736/800): Mass Spectrometry and the

  14. Imaging Mass Spectrometry in Neuroscience

    PubMed Central

    2013-01-01

    Imaging mass spectrometry is an emerging technique of great potential for investigating the chemical architecture in biological matrices. Although the potential for studying neurobiological systems is evident, the relevance of the technique for application in neuroscience is still in its infancy. In the present Review, a principal overview of the different approaches, including matrix assisted laser desorption ionization and secondary ion mass spectrometry, is provided with particular focus on their strengths and limitations for studying different neurochemical species in situ and in vitro. The potential of the various approaches is discussed based on both fundamental and biomedical neuroscience research. This Review aims to serve as a general guide to familiarize the neuroscience community and other biomedical researchers with the technique, highlighting its great potential and suitability for comprehensive and specific chemical imaging. PMID:23530951

  15. MASS SPECTROMETRY-BASED METABOLOMICS

    PubMed Central

    Dettmer, Katja; Aronov, Pavel A.; Hammock, Bruce D.

    2007-01-01

    This review presents an overview of the dynamically developing field of mass spectrometry-based metabolomics. Metabolomics aims at the comprehensive and quantitative analysis of wide arrays of metabolites in biological samples. These numerous analytes have very diverse physico-chemical properties and occur at different abundance levels. Consequently, comprehensive metabolomics investigations are primarily a challenge for analytical chemistry and specifically mass spectrometry has vast potential as a tool for this type of investigation. Metabolomics require special approaches for sample preparation, separation, and mass spectrometric analysis. Current examples of those approaches are described in this review. It primarily focuses on metabolic fingerprinting, a technique that analyzes all detectable analytes in a given sample with subsequent classification of samples and identification of differentially expressed metabolites, which define the sample classes. To perform this complex task, data analysis tools, metabolite libraries, and databases are required. Therefore, recent advances in metabolomics bioinformatics are also discussed. PMID:16921475

  16. Instrumentation for mass spectrometry: 1997

    SciTech Connect

    McLuckey, S.A.

    1997-08-01

    All mass spectrometry experiments involve the manipulation of material, an interface with the mass spectrometer, ionization, ion manipulation/analysis, detection and data collection/reduction. Each of these elements involve instrumentation. The wide range of species now amenable to mass spectrometry and the diverse areas of physical science in which it plays a role have led to a seemingly unlimited array of instrumental combinations. However, only a limited number of mass analyzers, and their combinations, dominate. The dominant analyzers include time-of-flight, Fourier transform ion cyclotron resonance, the Paul trap, the mass filter, and the sector mass spectrometer. Why there are so few (or so many, depending upon one`s point of view) can be understood upon consideration of a set of mass analyzer figures of merit. These include mass resolution, mass accuracy, mass range, dynamic range, abundance sensitivity, precision, efficiency, speed, MS{sup n} capability, compatibility with the ionizer, cost, and size. The most appropriate form of mass spectrometry is determined by the priorities of the particular measurement placed on the various mass analyzer characteristics and the relative strengths of the analyzers in meeting the requirements. Each of the analyzer types has a unique set of figures of merit that makes it optimally suited for particular applications. This paper discusses these figures of merit, provides data illustrating recent developments for each analyzer type, and gives the figures of merit of each type of analyzer as they stand in 1997. 101 refs., 24 figs.

  17. Study on CCR5 analogs and affinity peptides.

    PubMed

    Wu, Yingping; Deng, Riqiang; Wu, Wenyan

    2012-03-01

    The G protein-coupled receptor of human chemokine receptor 5 (CCR5) is a key target in the human immunodeficiency virus (HIV) infection process due to its major involvement in binding to the HIV type 1 (HIV-1) envelope glycoprotein gp120 and facilitating virus entry into the cells. The identification of naturally occurring CCR5 mutations (especially CCR5 delta-32) has allowed us to address the CCR5 molecule as a promising target to prevent or resist HIV infection in vivo. To obtain high-affinity peptides that can be used to block CCR5, CCR5 analogs with high conformational similarity are required. In this study, two recombinant proteins named CCR5 N-Linker-E2 and CCR5 mN-E1-E2 containing the fragments of the CCR5 N-terminal, the first extracellular loop or the second extracellular loop are cloned from a full-length human CCR5 cDNA. The recombinant human CCR5 analogs with self-cleavage activity of the intein Mxe or Ssp in the vector pTwinI were then produced with a high-yield expression and purification system in Escherichia coli. Experiments of extracellular epitope-activity identification (such as immunoprecipitation and indirective/competitive enzyme-linked immunosorbent assay) confirmed the close similarity between the epitope activity of the CCR5 analogs and that of the natural CCR5, suggesting the applicability of the recombinant CCR5 analogs as antagonists of the chemokine ligands. Subsequent screening of high-affinity peptides from the phage random-peptides library acquired nine polypeptides, which could be used as CCR5 peptide antagonists. The CCR5 analogs and affinity peptides elucidated in this paper provide us with a basis for further study of the mechanism of inhibition of HIV-1 infection.

  18. Isotope shift in the sulfur electron affinity: Observation and theory

    SciTech Connect

    Carette, Thomas; Scharf, Oliver; Godefroid, Michel; Froese Fischer, Charlotte

    2010-04-15

    The sulfur electron affinities {sup e}A(S) are measured by photodetachment microscopy for the two isotopes {sup 32}S and {sup 34}S (16 752.975 3(41) and 16 752.977 6(85) cm{sup -1}, respectively). The isotope shift in the electron affinity is found to be more probably positive, {sup e}A({sup 34}S)- {sup e}A({sup 32}S) =+0.0023(70) cm{sup -1}, but the uncertainty allows for the possibility that it may be either ''normal''[{sup e}A({sup 34}S) > {sup e}A({sup 32}S)] or ''anomalous''[{sup e}A({sup 34}S) < {sup e}A({sup 32}S)]. The isotope shift is estimated theoretically using elaborate correlation models, monitoring the electron affinity and the mass polarization term expectation value. The theoretical analysis predicts a very large specific mass shift (SMS) that counterbalances the normal mass shift (NMS) and produces an anomalous isotope shift {sup e}A({sup 34}S)- {sup e}A({sup 32}S) =-0.0053(24) cm{sup -1}, field shift corrections included. The total isotope shift can always be written as the sum of the NMS (here +0.0169 cm{sup -1}) and a residual isotope shift (RIS). Since the NMS has nearly no uncertainty, the comparison between experimental and theoretical RIS is more fair. With respective values of -0.0146(70) cm{sup -1} and -0.0222(24) cm{sup -1}, these residual isotope shifts are found to agree within the estimated uncertainties.

  19. The affinity of magnetic microspheres for Schistosoma eggs.

    PubMed

    Candido, Renata R F; Favero, Vivian; Duke, Mary; Karl, Stephan; Gutiérrez, Lucía; Woodward, Robert C; Graeff-Teixeira, Carlos; Jones, Malcolm K; St Pierre, Timothy G

    2015-01-01

    Schistosomiasis is a chronic parasitic disease of humans, with two species primarily causing the intestinal infection: Schistosoma mansoni and Schistosoma japonicum. Traditionally, diagnosis of schistosomiasis is achieved through direct visualisation of eggs in faeces using techniques that lack the sensitivity required to detect all infections, especially in areas of low endemicity. A recently developed method termed Helmintex™ is a very sensitive technique for detection of Schistosoma eggs and exhibits 100% sensitivity at 1.3 eggs per gram of faeces, enough to detect even low-level infections. The Helminthex™ method is based on the interaction of magnetic microspheres and schistosome eggs. Further understanding the underlying egg-microsphere interactions would enable a targeted optimisation of egg-particle binding and may thus enable a significant improvement of the Helmintex™ method and diagnostic sensitivity in areas with low infection rates. We investigated the magnetic properties of S. mansoni and S. japonicum eggs and their interactions with microspheres with different magnetic properties and surface functionalization. Eggs of both species exhibited higher binding affinity to the magnetic microspheres than the non-magnetic microspheres. Binding efficiency was further enhanced if the particles were coated with streptavidin. Schistosoma japonicum eggs bound more microspheres compared with S. mansoni. However, distinct differences within eggs of each species were also observed when the distribution of the number of microspheres bound per egg was modelled with double Poisson distributions. Using this approach, both S. japonicum and S. mansoni eggs fell into two groups, one having greater affinity for magnetic microspheres than the other, indicating that not all eggs of a species exhibit the same binding affinity. Our observations suggest that interaction between the microspheres and eggs is more likely to be related to surface charge-based electrostatic

  20. Surface affinity role in graphoepitaxy of lamellar block copolymers

    NASA Astrophysics Data System (ADS)

    Claveau, Guillaume; Quemere, Patrick; Argoud, Maxime; Hazart, Jerome; Barros, Patricia Pimenta; Sarrazin, Aurelien; Posseme, Nicolas; Tiron, Raluca; Chevalier, Xavier; Nicolet, Celia; Navarro, Christophe

    2016-07-01

    Overcoming the optical limitations of 193-nm immersion lithography can be achieved using directed self-assembly (DSA) of block-copolymers (BCPs) as a low-cost and versatile complementary technique. The goal of this paper is to investigate the potential of DSA to address line and space (L/S) high-resolution patterning by performing the density multiplication of lines with the graphoepitaxy approach. As surface affinity is a key parameter in self-assembly, three variations, or "flavors," of DSA template affinity are investigated regarding several success criteria such as morphology control or defectivity. More precisely, both the methodology to register DSA defects and the impact of process parameters on defectivity are detailed. Using the 300-mm pilot line available in LETI and Arkema's advanced materials, we investigate process optimization of DSA line/space patterning of a 38-nm period lamellar PS-b-PMMA BCP (L38). Our integration scheme is based on BCP self-assembly inside organic hard mask guiding patterns obtained using 193i-nm lithography. Defect analysis coupled with the fine tuning of process parameters (annealing, brush material) provided the optimum conditions for the L38 self-assembly. Using such conditions, DSA using the three affinity flavors is investigated by means of SEM top-view and cross-section review. Lithographic performances of one selected flavor are then evaluated with the comparison of process windows function of either commensurability, morphology, or roughness. This work is meant as a guideline for the graphoepitaxy optimization of materials and process parameters on a 300-mm platform.

  1. Affine transformations from aerial photos to computer compatible tapes

    NASA Technical Reports Server (NTRS)

    Peet, F. G.; Mack, A. R.; Crosson, L. S.

    1974-01-01

    During the development of a project to estimate wheat production, it became necessary to pull data, corresponding to particular fields in a test site, off an ERTS computer compatible tape. Aerial photographs and topographic maps were on hand for the test site. A method was devised, using an affine transformation, to relate the aerial photographs or topographic maps to the tapes. One can thereby access data on the tape corresponding to regions covered by only a few pixels. The theory can be used for the registration of two tapes for the same area and for the geometric correction of images.

  2. Nine switch-affine neurons suffice for Turing universality.

    PubMed

    Siegelmann, H T.; Margenstern, M

    1999-06-01

    In a previous work Pollack showed that a particular type of heterogeneous processor network is Turing universal. Siegelmann and Sontag (1991) showed the universality of homogeneous networks of first-order neurons having piecewise-linear activation functions. Their result was generalized by Kilian and Siegelmann (1996) to include various sigmoidal activation functions. Here we focus on a type of high-order neurons called switch-affine neurons, with piecewise-linear activation functions, and prove that nine such neurons suffice for simulating universal Turing machines.

  3. Friction and plasticity between self-affine surfaces

    NASA Astrophysics Data System (ADS)

    Luan, Binquan; Robbins, Mark; Harrison, Judith

    2006-03-01

    Simulations are used to study the contact area and adhesion between two amorphous solids with self-affine fractal surfaces, and the results are compared to continuum calculations. The friction between non-adhesive surfaces is proportional to load, but the coefficient of friction increases with roughness. The friction is much higher than expected for elasticallly deforming surfaces,^* and substantial plastic deformation is observed. Indeed, friction forces for different surface roughness collapse when plotted against the number of plastic rearrangements per unit sliding distance. Including adhesion leads to an increase in both friction and plasticity. ^* M. H. Müser, L. Wenning, and M. O. Robbins, Phys. Rev. Lett. 86, 1295 (2001).

  4. An affine projection algorithm using grouping selection of input vectors

    NASA Astrophysics Data System (ADS)

    Shin, JaeWook; Kong, NamWoong; Park, PooGyeon

    2011-10-01

    This paper present an affine projection algorithm (APA) using grouping selection of input vectors. To improve the performance of conventional APA, the proposed algorithm adjusts the number of the input vectors using two procedures: grouping procedure and selection procedure. In grouping procedure, the some input vectors that have overlapping information for update is grouped using normalized inner product. Then, few input vectors that have enough information for for coefficient update is selected using steady-state mean square error (MSE) in selection procedure. Finally, the filter coefficients update using selected input vectors. The experimental results show that the proposed algorithm has small steady-state estimation errors comparing with the existing algorithms.

  5. The local electron affinity for non-minimal basis sets.

    PubMed

    Clark, Timothy

    2010-07-01

    A technique known as intensity filtering is introduced to select valence-like virtual orbitals for calculating the local electron affinity, EA(L). Intensity filtering allows EA(L) to be calculated using semiempirical molecular orbital techniques that include polarisation functions. Without intensity filtering, such techniques yield spurious EA(L) values that are dominated by the polarisation functions. As intensity filtering should also be applicable for ab initio or density functional theory calculations with large basis sets, it also makes EA(L) available for these techniques.

  6. Statistical geometric affinity in human brain electric activity

    NASA Astrophysics Data System (ADS)

    Chornet-Lurbe, A.; Oteo, J. A.; Ros, J.

    2007-05-01

    The representation of the human electroencephalogram (EEG) records by neurophysiologists demands standardized time-amplitude scales for their correct conventional interpretation. In a suite of graphical experiments involving scaling affine transformations we have been able to convert electroencephalogram samples corresponding to any particular sleep phase and relaxed wakefulness into each other. We propound a statistical explanation for that finding in terms of data collapse. As a sequel, we determine characteristic time and amplitude scales and outline a possible physical interpretation. An analysis for characteristic times based on lacunarity is also carried out as well as a study of the synchrony between left and right EEG channels.

  7. Affine reflection groups for tiling applications: Knot theory and DNA

    NASA Astrophysics Data System (ADS)

    Bodner, M.; Patera, J.; Peterson, M.

    2012-01-01

    We present in this paper some non-conventional applications of affine Weyl groups Waff of rank 2, the symmetry group of the tiling/lattice. We first develop and present the tools for applications requiring tilings of a real Euclidean plane {R}^2. We then elucidate the equivalence of these tilings with 2D projections of knots. The resulting mathematical structure provides a framework within which is encompassed recent work utilizing knot theory for modeling the structure and function of genetic molecules, specifically the action of particular enzymes in altering the topology of DNA in site-specific recombination.

  8. Affinity capture using peptide-functionalized magnetic nanoparticles to target Staphylococcus aureus

    NASA Astrophysics Data System (ADS)

    Kuo, Fang-Yin; Lin, Wei-Lien; Chen, Yu-Chie

    2016-04-01

    Staphylococcus aureus, a commonly found pathogen, can cause food poisoning and infections. Thus, it is necessary to develop analytical methods for the rapid screening of S. aureus in suspicious samples. Magnetic nanoparticles (MNPs) are widely used as affinity probes to selectively enrich target species from complex samples because of their high specific surface area and magnetic properties. The MNP surface should be functionalized to have the capability to target specific species. We herein propose a straightforward method to functionalize aluminum oxide-coated iron oxide (Fe3O4@Al2O3) MNPs with the peptide HHHHHHDEEGLFVD (D). The peptide D was comprised of three domains: polyhistidine (H6) used as the linker, DEE added as the spacer, and GLFVD used for targeting S. aureus. D was immobilized on the surface of Fe3O4@Al2O3 MNPs through H6-Al chelation. Our results showed that the D-functionalized Fe3O4@Al2O3 MNPs (D-Fe3O4 MNPs) possess the capability to target S. aureus. The selective trapping experiments were conducted under microwave-heating for only 60 s, and sufficient bacterial cells were trapped by the MNPs to be identified by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We demonstrated that the D-Fe3O4@Al2O3 MNPs combined with MALDI-MS can be used to rapidly characterize trace amounts of S. aureus in complex juice and egg samples.Staphylococcus aureus, a commonly found pathogen, can cause food poisoning and infections. Thus, it is necessary to develop analytical methods for the rapid screening of S. aureus in suspicious samples. Magnetic nanoparticles (MNPs) are widely used as affinity probes to selectively enrich target species from complex samples because of their high specific surface area and magnetic properties. The MNP surface should be functionalized to have the capability to target specific species. We herein propose a straightforward method to functionalize aluminum oxide-coated iron oxide (Fe3O4@Al2O3) MNPs with the

  9. Linear algorithms of affine synthesis in the Lebesgue space L^1 \\lbrack 0,1 \\rbrack

    NASA Astrophysics Data System (ADS)

    Terekhin, Pavel A.

    2010-10-01

    We prove that there are no linear algorithms of affine synthesis for affine systems in the Lebesgue space L^1 \\lbrack 0,1 \\rbrack with respect to the model space \\ell^1, although the corresponding affine synthesis problem has a positive solution under the most general assumptions. At the same time, by imposing additional conditions on the generating function of the affine system, we can give an explicit linear algorithm of affine synthesis in the Lebesgue space when the model space is that of the coefficients of the system. This linear algorithm generalizes the Fourier-Haar expansion into orthogonal series.

  10. [Progresses in screening active compounds from herbal medicine by affinity chromatography].

    PubMed

    Feng, Ying-shu; Tong, Shan-shan; Xu, Xi-ming; Yu, Jiang-nan

    2015-03-01

    Affinity chromatography is a chromatographic method for separating molecules using the binding characteristics of the stationary phase with potential drug molecules. This method can be performed as a high throughput screening method and a chromatographic separation method to screen a variety of active drugs. This paper summarizes the history of affinity chromatography, screening technology of affinity chromatography, and application of affinity chromatography in screening bio-active compounds in herbal medicines, and then discusses its application prospects, in order to broaden applications of the affinity chromatography in drug screening.

  11. Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124

    SciTech Connect

    Auld, Douglas S.; Lovell, Scott; Thorne, Natasha; Lea, Wendy A.; Maloney, David J.; Shen, Min; Rai, Ganesha; Battaile, Kevin P.; Thomas, Craig J.; Simeonov, Anton; Hanzlik, Robert P.; Inglese, James

    2010-04-07

    Firefly luciferase (FLuc), an ATP-dependent bioluminescent reporter enzyme, is broadly used in chemical biology and drug discovery assays. PTC124 Ataluren; (3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid) discovered in an FLuc-based assay targeting nonsense codon suppression, is an unusually potent FLuc-inhibitor. Paradoxically, PTC124 and related analogs increase cellular FLuc activity levels by posttranslational stabilization. In this study, we show that FLuc inhibition and stabilization is the result of an inhibitory product formed during the FLuc-catalyzed reaction between its natural substrate, ATP, and PTC124. A 2.0 {angstrom} cocrystal structure revealed the inhibitor to be the acyl-AMP mixed-anhydride adduct PTC124-AMP, which was subsequently synthesized and shown to be a high-affinity multisubstrate adduct inhibitor (MAI; KD = 120 pM) of FLuc. Biochemical assays, liquid chromatography/mass spectrometry, and near-attack conformer modeling demonstrate that formation of this novel MAI is absolutely dependent upon the precise positioning and reactivity of a key meta-carboxylate of PTC124 within the FLuc active site. We also demonstrate that the inhibitory activity of PTC124-AMP is relieved by free coenzyme A, a component present at high concentrations in luciferase detection reagents used for cell-based assays. This explains why PTC124 can appear to increase, instead of inhibit, FLuc activity in cell-based reporter gene assays. To our knowledge, this is an unusual example in which the 'off-target' effect of a small molecule is mediated by an MAI mechanism.

  12. Geochemical Evidence of the Seasonality, Affinity and Pigmenation of Solenopora jurassica

    SciTech Connect

    Barden, Holly E.; Behnsen, Julia; Bergmann, Uwe; Leng, Melanie J.; Manning, Phillip L.; Withers, Philip J.; Wogelius, Roy A.; van Dongen, Bart E.; Silva, Lucas C. R.

    2015-09-14

    Solenopora jurassica is a fossil calcareous alga that functioned as an important reef-building organism during the Palaeozoic. It is of significant palaeobiological interest due to its distinctive but poorly understood pink and white banding. Though widely accepted as an alga there is still debate over its taxonomic affinity, with recent work arguing that it should be reclassified as a chaetetid sponge. The banding is thought to be seasonal, but there is no conclusive evidence for this. Other recent work has, however demonstrated the presence of a unique organic boron-containing pink/red pigment in the pink bands of S. jurassica. We present new geochemical evidence concerning the seasonality and pigmentation of S. jurassica. Seasonal growth cycles are demonstrated by X-ray radiography, which shows differences in calcite density, and by varying δ13C composition of the bands. Temperature variation in the bands is difficult to constrain accurately due to conflicting patterns arising from Mg/Ca molar ratios and δ18O data. Fluctuating chlorine levels indicate increased salinity in the white bands, when combined with the isotope data this suggests more suggestive of marine conditions during formation of the white band and a greater freshwater component (lower chlorinity) during pink band precipitation (δ18O). Increased photosynthesis is inferred within the pink bands in comparison to the white, based on δ13C. Pyrolysis Gas Chromatography Mass Spectrometry (Py-GCMS) and Fourier Transform Infrared Spectroscopy (FTIR) show the presence of tetramethyl pyrrole, protein moieties and carboxylic acid groups, suggestive of the presence of the red algal pigment phycoerythrin. This is consistent with the pink colour of S. jurassica. As phycoerythrin is only known to occur in algae and cyanobacteria, and no biomarker evidence of bacteria or sponges was detected we conclude S. jurassica is most likely an alga. Pigment analysis may be a

  13. Applicability of tandem affinity purification MudPIT to pathway proteomics in yeast.

    PubMed

    Graumann, Johannes; Dunipace, Leslie A; Seol, Jae Hong; McDonald, W Hayes; Yates, John R; Wold, Barbara J; Deshaies, Raymond J

    2004-03-01

    A combined multidimensional chromatography-mass spectrometry approach known as "MudPIT" enables rapid identification of proteins that interact with a tagged bait while bypassing some of the problems associated with analysis of polypeptides excised from SDS-polyacrylamide gels. However, the reproducibility, success rate, and applicability of MudPIT to the rapid characterization of dozens of proteins have not been reported. We show here that MudPIT reproducibly identified bona fide partners for budding yeast Gcn5p. Additionally, we successfully applied MudPIT to rapidly screen through a collection of tagged polypeptides to identify new protein interactions. Twenty-five proteins involved in transcription and progression through mitosis were modified with a new tandem affinity purification (TAP) tag. TAP-MudPIT analysis of 22 yeast strains that expressed these tagged proteins uncovered known or likely interacting partners for 21 of the baits, a figure that compares favorably with traditional approaches. The proteins identified here comprised 102 previously known and 279 potential physical interactions. Even for the intensively studied Swi2p/Snf2p, the catalytic subunit of the Swi/Snf chromatin remodeling complex, our analysis uncovered a new interacting protein, Rtt102p. Reciprocal tagging and TAP-MudPIT analysis of Rtt102p revealed subunits of both the Swi/Snf and RSC complexes, identifying Rtt102p as a common interactor with, and possible integral component of, these chromatin remodeling machines. Our experience indicates it is feasible for an investigator working with a single ion trap instrument in a conventional molecular/cellular biology laboratory to carry out proteomic characterization of a pathway, organelle, or process (i.e. "pathway proteomics") by systematic application of TAP-MudPIT.

  14. Geochemical Evidence of the Seasonality, Affinity and Pigmenation of Solenopora jurassica

    PubMed Central

    Barden, Holly E.; Behnsen, Julia; Bergmann, Uwe; Leng, Melanie J.; Manning, Phillip L.; Withers, Philip J.; Wogelius, Roy A.; van Dongen, Bart E.

    2015-01-01

    Solenopora jurassica is a fossil calcareous alga that functioned as an important reef-building organism during the Palaeozoic. It is of significant palaeobiological interest due to its distinctive but poorly understood pink and white banding. Though widely accepted as an alga there is still debate over its taxonomic affinity, with recent work arguing that it should be reclassified as a chaetetid sponge. The banding is thought to be seasonal, but there is no conclusive evidence for this. Other recent work has, however demonstrated the presence of a unique organic boron-containing pink/red pigment in the pink bands of S. jurassica. We present new geochemical evidence concerning the seasonality and pigmentation of S. jurassica. Seasonal growth cycles are demonstrated by X-ray radiography, which shows differences in calcite density, and by varying δ13C composition of the bands. Temperature variation in the bands is difficult to constrain accurately due to conflicting patterns arising from Mg/Ca molar ratios and δ18O data. Fluctuating chlorine levels indicate increased salinity in the white bands, when combined with the isotope data this suggests more suggestive of marine conditions during formation of the white band and a greater freshwater component (lower chlorinity) during pink band precipitation (δ18O). Increased photosynthesis is inferred within the pink bands in comparison to the white, based on δ13C. Pyrolysis Gas Chromatography Mass Spectrometry (Py-GCMS) and Fourier Transform Infrared Spectroscopy (FTIR) show the presence of tetramethyl pyrrole, protein moieties and carboxylic acid groups, suggestive of the presence of the red algal pigment phycoerythrin. This is consistent with the pink colour of S. jurassica. As phycoerythrin is only known to occur in algae and cyanobacteria, and no biomarker evidence of bacteria or sponges was detected we conclude S. jurassica is most likely an alga. Pigment analysis may be a reliable classification method for fossil

  15. Identification of Protein Partners in Mycobacteria Using a Single-Step Affinity Purification Method

    PubMed Central

    Cysewski, Dominik; Stoduś, Krystian; Kowalska, Katarzyna; Dziembowski, Andrzej

    2014-01-01

    Tuberculosis is a leading cause of death in developing countries. Efforts are being made to both prevent its spread and improve curability rates. Understanding the biology of the bacteria causing the disease, Mycobacterium tuberculosis (M. tuberculosis), is thus vital. We have implemented improved screening methods for protein–protein interactions based on affinity purification followed by high-resolution mass spectrometry. This method can be efficiently applied to both medium- and high-throughput studies aiming to characterize protein–protein interaction networks of tubercle bacilli. Of the 4 tested epitopes FLAG, enhanced green fluorescent protein (eGFP), protein A and haemagglutinin, the eGFP tag was found to be most useful on account of its easily monitored expression and its ability to function as a simultaneous tool for subcellular localization studies. It presents a relatively low background with cost-effective purification. RNA polymerase subunit A (RpoA) was used as a model for investigation of a large protein complex. When used as bait, it co-purified with all remaining RNA polymerase core subunits as well as many accessory proteins. The amount of RpoA strongly correlated with the amount of quantification peptide used as part of the tagging system in this study (SH), making it applicable for semi-quantification studies. Interactions between the components of the RpoA-eGFP protein complex were further confirmed using protein cross-linking. Dynamic changes in the composition of protein complexes under induction of UV damage were observed when UvrA-eGFP expressing cells treated with UV light were used to co-purify UvrA interaction partners. PMID:24664103

  16. Geochemical Evidence of the Seasonality, Affinity and Pigmenation of Solenopora jurassica

    DOE PAGES

    Barden, Holly E.; Behnsen, Julia; Bergmann, Uwe; ...

    2015-09-14

    Solenopora jurassica is a fossil calcareous alga that functioned as an important reef-building organism during the Palaeozoic. It is of significant palaeobiological interest due to its distinctive but poorly understood pink and white banding. Though widely accepted as an alga there is still debate over its taxonomic affinity, with recent work arguing that it should be reclassified as a chaetetid sponge. The banding is thought to be seasonal, but there is no conclusive evidence for this. Other recent work has, however demonstrated the presence of a unique organic boron-containing pink/red pigment in the pink bands of S. jurassica. We presentmore » new geochemical evidence concerning the seasonality and pigmentation of S. jurassica. Seasonal growth cycles are demonstrated by X-ray radiography, which shows differences in calcite density, and by varying δ13C composition of the bands. Temperature variation in the bands is difficult to constrain accurately due to conflicting patterns arising from Mg/Ca molar ratios and δ18O data. Fluctuating chlorine levels indicate increased salinity in the white bands, when combined with the isotope data this suggests more suggestive of marine conditions during formation of the white band and a greater freshwater component (lower chlorinity) during pink band precipitation (δ18O). Increased photosynthesis is inferred within the pink bands in comparison to the white, based on δ13C. Pyrolysis Gas Chromatography Mass Spectrometry (Py-GCMS) and Fourier Transform Infrared Spectroscopy (FTIR) show the presence of tetramethyl pyrrole, protein moieties and carboxylic acid groups, suggestive of the presence of the red algal pigment phycoerythrin. This is consistent with the pink colour of S. jurassica. As phycoerythrin is only known to occur in algae and cyanobacteria, and no biomarker evidence of bacteria or sponges was detected we conclude S. jurassica is most likely an alga. Pigment analysis may be a reliable classification method for

  17. Identification of novel surface-exposed proteins of Rickettsia rickettsii by affinity purification and proteomics.

    PubMed

    Gong, Wenping; Xiong, Xiaolu; Qi, Yong; Jiao, Jun; Duan, Changsong; Wen, Bohai

    2014-01-01

    Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is the most pathogenic member among Rickettsia spp. Surface-exposed proteins (SEPs) of R. rickettsii may play important roles in its pathogenesis or immunity. In this study, R. rickettsii organisms were surface-labeled with sulfo-NHS-SS-biotin and the labeled proteins were affinity-purified with streptavidin. The isolated proteins were separated by two-dimensional electrophoresis, and 10 proteins were identified among 23 protein spots by electrospray ionization tandem mass spectrometry. Five (OmpA, OmpB, GroEL, GroES, and a DNA-binding protein) of the 10 proteins were previously characterized as surface proteins of R. rickettsii. Another 5 proteins (Adr1, Adr2, OmpW, Porin_4, and TolC) were first recognized as SEPs of R. rickettsii herein. The genes encoding the 5 novel SEPs were expressed in Escherichia coli cells, resulting in 5 recombinant SEPs (rSEPs), which were used to immunize mice. After challenge with viable R. rickettsii cells, the rickettsial load in the spleen, liver, or lung of mice immunized with rAdr2 and in the lungs of mice immunized with other rSEPs excluding rTolC was significantly lower than in mice that were mock-immunized with PBS. The in vitro neutralization test revealed that sera from mice immunized with rAdr1, rAdr2, or rOmpW reduced R. rickettsii adherence to and invasion of vascular endothelial cells. The immuno-electron microscopic assay clearly showed that the novel SEPs were located in the outer and/or inner membrane of R. rickettsii. Altogether, the 5 novel SEPs identified herein might be involved in the interaction of R. rickettsii with vascular endothelial cells, and all of them except TolC were protective antigens.

  18. Experimental and theoretical binding affinity between polyvinylpolypyrrolidone and selected phenolic compounds from food matrices.

    PubMed

    Durán-Lara, Esteban F; López-Cortés, Xaviera A; Castro, Ricardo I; Avila-Salas, Fabián; González-Nilo, Fernando D; Laurie, V Felipe; Santos, Leonardo S

    2015-02-01

    Polyvinylpolypyrrolidone (PVPP) is a fining agent, widely used in winemaking and brewing, whose mode of action in removing phenolic compounds has not been fully characterised. The aim of this study was to evaluate the experimental and theoretical binding affinity of PVPP towards six phenolic compounds representing different types of phenolic species. The interaction between PVPP and phenolics was evaluated in model solutions, where hydroxyl groups, hydrophobic bonding and steric hindrance were characterised. The results of the study indicated that PVPP exhibits high affinity for quercetin and catechin, moderate affinity for epicatechin, gallic acid and lower affinity for 4-methylcatechol and caffeic acid. The affinity has a direct correlation with the hydroxylation degree of each compound. The results show that the affinity of PVPP towards phenols is related with frontier orbitals. This work demonstrates a direct correlation between the experimental affinity and the interaction energy calculations obtained through computational chemistry methods.

  19. Bicarbonate-form anion exchange: affinity, regeneration, and stoichiometry.

    PubMed

    Rokicki, Christopher A; Boyer, Treavor H

    2011-01-01

    Magnetic ion exchange (MIEX) is an effective process for removing dissolved organic carbon (DOC) from natural waters, but its implementation has been limited due to production of waste sodium chloride solution (i.e., brine) from the regeneration process. Chloride is of concern because elevated concentrations can have adverse effects on engineered and natural systems. The goal of this research was to explore the efficacy of using anion exchange resin with bicarbonate as the mobile counter ion, which would produce a non-chloride regeneration solution. It was found that bicarbonate-form MIEX resin had a similar affinity as chloride-form MIEX resin for sulfate, nitrate, DOC, and ultraviolet-absorbing substances. Both bicarbonate-form and chloride-form MIEX resins showed the greatest removal efficiencies as fresh resin, and removal efficiency decreased with multiple regeneration cycles. Nevertheless, sodium bicarbonate solution was as effective as sodium chloride solution at regenerating MIEX resin. Regeneration of the bicarbonate-form MIEX resin was illustrated by sparging carbon dioxide gas in a water/resin slurry. This regeneration process would eliminate the need for the addition of salts such as sodium chloride or sodium bicarbonate. The stoichiometry of the bicarbonate-form resin revealed that the bicarbonate was deprotonating within the resin matrix leading to a mixture of both carbonate and bicarbonate mobile counter ions. This work makes an important contribution to ion exchange applications for water treatment by evaluating the affinity, regeneration, and stoichiometry of bicarbonate-form anion exchange.

  20. Flexible Molybdenum Electrodes towards Designing Affinity Based Protein Biosensors

    PubMed Central

    Kamakoti, Vikramshankar; Panneer Selvam, Anjan; Radha Shanmugam, Nandhinee; Muthukumar, Sriram; Prasad, Shalini

    2016-01-01

    Molybdenum electrode based flexible biosensor on porous polyamide substrates has been fabricated and tested for its functionality as a protein affinity based biosensor. The biosensor performance was evaluated using a key cardiac biomarker; cardiac Troponin-I (cTnI). Molybdenum is a transition metal and demonstrates electrochemical behavior upon interaction with an electrolyte. We have leveraged this property of molybdenum for designing an affinity based biosensor using electrochemical impedance spectroscopy. We have evaluated the feasibility of detection of cTnI in phosphate-buffered saline (PBS) and human serum (HS) by measuring impedance changes over a frequency window from 100 mHz to 1 MHz. Increasing changes to the measured impedance was correlated to the increased dose of cTnI molecules binding to the cTnI antibody functionalized molybdenum surface. We achieved cTnI detection limit of 10 pg/mL in PBS and 1 ng/mL in HS medium. The use of flexible substrates for designing the biosensor demonstrates promise for integration with a large-scale batch manufacturing process. PMID:27438863

  1. Expression and affinity purification of recombinant proteins from plants

    NASA Technical Reports Server (NTRS)

    Desai, Urvee A.; Sur, Gargi; Daunert, Sylvia; Babbitt, Ruth; Li, Qingshun

    2002-01-01

    With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, beta-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system. Copyright 2002 Elsevier Science (USA).

  2. The Mobile Phone Affinity Scale: Enhancement and Refinement

    PubMed Central

    Rosen, Rochelle K

    2016-01-01

    Background Existing instruments that assess individuals’ relationships with mobile phones tend to focus on negative constructs such as addiction or dependence, and appear to assume that high mobile phone use reflects pathology. Mobile phones can be beneficial for health behavior change, disease management, work productivity, and social connections, so there is a need for an instrument that provides a more balanced assessment of the various aspects of individuals’ relationships with mobile phones. Objective The purpose of this research was to develop, revise, and validate the Mobile Phone Affinity Scale, a multi-scale instrument designed to assess key factors associated with mobile phone use. Methods Participants (N=1058, mean age 33) were recruited from Amazon Mechanical Turk between March and April of 2016 to complete a survey that assessed participants’ mobile phone attitudes and use, anxious and depressive symptoms, and resilience. Results Confirmatory factor analysis supported a 6-factor model. The final measure consisted of 24 items, with 4 items on each of 6 factors: Connectedness, Productivity, Empowerment, Anxious Attachment, Addiction, and Continuous Use. The subscales demonstrated strong internal consistency (Cronbach alpha range=0.76-0.88, mean 0.83), and high item factor loadings (range=0.57-0.87, mean 0.75). Tests for validity further demonstrated support for the individual subscales. Conclusions Mobile phone affinity may have an important impact in the development and effectiveness of mobile health interventions, and continued research is needed to assess its predictive ability in health behavior change interventions delivered via mobile phones. PMID:27979792

  3. Compensating Enthalpic and Entropic Changes Hinder Binding Affinity Optimization

    SciTech Connect

    Lafont,V.; Armstrong, A.; Ohtaka, H.; Kiso, Y.; Amzel, L.; Freire, E.

    2007-01-01

    A common strategy to improve the potency of drug candidates is to introduce chemical functionalities, like hydrogen bond donors or acceptors, at positions where they are able to establish strong interactions with the target. However, it is often observed that the added functionalities do not necessarily improve potency even if they form strong hydrogen bonds. Here, we explore the thermodynamic and structural basis for those observations. KNI-10033 is a potent experimental HIV-1 protease inhibitor with picomolar affinity against the wild-type enzyme (Kd = 13 pm). The potency of the inhibitor is the result of favorable enthalpic (?H = -8.2 kcal/mol) and entropic (-T?S = -6.7 kcal/mol) interactions. The replacement of the thioether group in KNI-10033 by a sulfonyl group (KNI-10075) results in a strong hydrogen bond with the amide of Asp 30B of the HIV-1 protease. This additional hydrogen bond improves the binding enthalpy by 3.9 kcal/mol; however, the enthalpy gain is completely compensated by an entropy loss, resulting in no affinity change. Crystallographic and thermodynamic analysis of the inhibitor/protease complexes indicates that the entropy losses are due to a combination of conformational and solvation effects. These results provide a set of practical guidelines aimed at overcoming enthalpy/entropy compensation and improve binding potency.

  4. Boronate affinity adsorption of RNA: possible role of conformational changes

    NASA Technical Reports Server (NTRS)

    Singh, N.; Willson, R. C.; Fox, G. E. (Principal Investigator)

    1999-01-01

    Batch equilibrium adsorption isotherm determination is used to characterize the adsorption of mixed yeast RNA on agarose-immobilized m-aminophenylboronic acid. It is shown that the affinity-enhancing influence of divalent cations depends strongly on the precise nature of the cation used, with barium being far more effective than the conventionally-used magnesium. This adsorption-promoting influence of barium is suggested to arise primarily from ionic influences on the structure and rigidity of the RNA molecule, as the adsorption of ribose-based small molecules is not similarly affected. The substitution of barium for the standard magnesium counterion does not greatly promote the adsorption of DNA, implying that the effect is specific to RNA and may be useful in boronate-based RNA separations. RNA adsorption isotherms exhibit sharp transitions as functions of temperature, and these transitions occur at different temperatures with Mg2+ and with Ba2+. Adsorption affinity and capacity were found to increase markedly at lower temperatures, suggestive of an enthalpically favored interaction process. The stoichiometric displacement parameter, Z, in Ba2+ buffer is three times the value in Mg2+ buffer, and is close to unity.

  5. 01-ERD-111 - The Development of Synthetic High Affinity Ligands

    SciTech Connect

    Perkins, J; Balhorn, R; Cosman, M; Lightstone, F; Zeller, L

    2004-02-05

    The aim of this project was to develop Synthetic High-Affinity Ligands (SHALs), which bind with high affinity and specificity to proteins of interest for national security and cancer therapy applications. The aim of producing synthetic ligands for sensory devices as an alternative to antibody-based detection assays and therapeutic agents is to overcome the drawbacks associated with antibody-based in next-generation sensors and systems. The focus area of the project was the chemical synthesis of the SHALs. The project concentrated on two different protein targets. (a) The C fragment of tetanus and botulinum toxin, potential biowarfare agents. A SHAL for tetanus or botulinum toxin would be incorporated into a sensory device for the toxins. (b) HLA-DR10, a protein found in high abundance on the surface of Non-Hodgkins Lymphoma. A SHAL specific to a tumor marker, labeled with a radionuclide, would enable the targeted delivery of radiation therapy to metastatic disease. The technical approach used to develop a SHAL for each protein target will be described in more detail below. However, in general, the development of a SHAL requires a combination of computational modeling techniques, modern nuclear magnetic resonance spectroscopy (NMR) and synthetic chemistry.

  6. Robust activation method for negative electron affinity photocathodes

    DOEpatents

    Mulhollan, Gregory A.; Bierman, John C.

    2011-09-13

    A method by which photocathodes(201), single crystal, amorphous, or otherwise ordered, can be surface modified to a robust state of lowered and in best cases negative, electron affinity has been discovered. Conventional methods employ the use of Cs(203) and an oxidizing agent(207), typically carried by diatomic oxygen or by more complex molecules, for example nitrogen trifluoride, to achieve a lowered electron affinity(404). In the improved activation method, a second alkali, other than Cs(205), is introduced onto the surface during the activation process, either by co-deposition, yo-yo, or sporadic or intermittent application. Best effect for GaAs photocathodes has been found through the use of Li(402) as the second alkali, though nearly the same effect can be found by employing Na(406). Suitable photocathodes are those which are grown, cut from boules, implanted, rolled, deposited or otherwise fabricated in a fashion and shape desired for test or manufacture independently supported or atop a support structure or within a framework or otherwise affixed or suspended in the place and position required for use.

  7. Affinity purification of copper chelating peptides from chickpea protein hydrolysates.

    PubMed

    Megías, Cristina; Pedroche, Justo; Yust, Maria M; Girón-Calle, Julio; Alaiz, Manuel; Millan, Francisco; Vioque, Javier

    2007-05-16

    Chickpea protein hydrolysates obtained with alcalase and flavourzyme were used for purification of copper chelating peptides by affinity chromatography using copper immobilized on solid supports. The chelating activity of purified peptides was indirectly measured by the inhibition of beta-carotene oxidation in the presence of copper. Two protein hydrolysates, obtained after 10 and 100 min of hydrolysis, were the most inhibitory of beta-carotene oxidation. Purified copper chelating peptides from these protein hydrolysates contained 19.7 and 35.1% histidine, respectively, in comparison to 2.7 and 2.6% in the protein hydrolysates. Chelating peptides from hydrolysate obtained after 10 min of hydrolysis were the most antioxidative being 8.3 times more antioxidative than the hydrolysate, while chelating peptides purified from protein hydrolysate obtained after 100 min were 3.1 times more antioxidative than its hydrolysate. However, the histidine content was higher in peptides derived from the 100 min hydrolysate (19.7 against 35.1% in 10 min hydrolysate), indicating that this amino acid is not the only factor involved in the antioxidative activity, and other factors such as peptide size or amino acid sequence are also determinant. This manuscript shows that affinity chromatography is a useful procedure for purification of copper chelating peptides. This method can be extended to other metals of interest in nutrition, such as calcium, iron, or zinc. Purified chelating peptides, in addition to their antioxidative properties, may also be useful in food mineral fortification for increasing the bioavailability of these metals.

  8. Self-affinity in the dengue fever time series

    NASA Astrophysics Data System (ADS)

    Azevedo, S. M.; Saba, H.; Miranda, J. G. V.; Filho, A. S. Nascimento; Moret, M. A.

    2016-06-01

    Dengue is a complex public health problem that is common in tropical and subtropical regions. This disease has risen substantially in the last three decades, and the physical symptoms depict the self-affine behavior of the occurrences of reported dengue cases in Bahia, Brazil. This study uses detrended fluctuation analysis (DFA) to verify the scale behavior in a time series of dengue cases and to evaluate the long-range correlations that are characterized by the power law α exponent for different cities in Bahia, Brazil. The scaling exponent (α) presents different long-range correlations, i.e. uncorrelated, anti-persistent, persistent and diffusive behaviors. The long-range correlations highlight the complex behavior of the time series of this disease. The findings show that there are two distinct types of scale behavior. In the first behavior, the time series presents a persistent α exponent for a one-month period. For large periods, the time series signal approaches subdiffusive behavior. The hypothesis of the long-range correlations in the time series of the occurrences of reported dengue cases was validated. The observed self-affinity is useful as a forecasting tool for future periods through extrapolation of the α exponent behavior. This complex system has a higher predictability in a relatively short time (approximately one month), and it suggests a new tool in epidemiological control strategies. However, predictions for large periods using DFA are hidden by the subdiffusive behavior.

  9. Impact of crystalline quality on neuronal affinity of pristine graphene.

    PubMed

    Veliev, Farida; Briançon-Marjollet, Anne; Bouchiat, Vincent; Delacour, Cécile

    2016-04-01

    Due to its outstanding mechanical and electrical properties as well as chemical inertness, graphene has attracted a growing interest in the field of bioelectric interfacing. Herein, we investigate the suitability of pristine, i.e. without a cell adhesive coating, chemical vapor deposition (CVD) grown monolayer graphene to act as a platform for neuronal growth. We study the development of primary hippocampal neurons grown on bare graphene (transferred on glass coverslip) for up to 5 days and show that pristine graphene significantly improves the neurons adhesion and outgrowth at the early stage of culture (1-2 days in vitro). At the later development stage, neurons grown on coating free graphene (untreated with poly-L-lysine) show remarkably well developed neuritic architecture similar to those cultured on conventional poly-L-lysine coated glass coverslips. This exceptional possibility to bypass the adhesive coating allows a direct electrical contact of graphene to the cells and reveals its great potential for chronic medical implants and tissue engineering. Moreover, regarding the controversial results obtained on the neuronal affinity of pristine graphene and its ability to support neuronal growth without the need of polymer or protein coating, we found that the crystallinity of CVD grown graphene plays an important role in neuronal attachment, outgrowth and axonal specification. In particular, we show that the decreasing crystalline quality of graphene tunes the neuronal affinity from highly adhesive to fully repellent.

  10. Purification of cytochrome c oxidase by lysine-affinity chromatography.

    PubMed

    Felsch, J; Kotake, S; Copeland, R A

    1992-02-01

    A method for the purification of cytochrome c oxidase that is based on the affinity of this enzyme for polycations such as poly-L-lysine is described. When detergent extracts of bovine cardiac mitochondria were applied to either a poly-L-lysine-agarose or a lysine-Sepharose column at low ionic strength, cytochrome c oxidase was found to adhere tightly, whereas the bulk of the proteins were eluted by washing with the same buffer. The cytochrome c oxidase was eluted by application of a linear potassium chloride gradient to the columns. The resulting enzyme was identical to that obtained by more traditional purification methods in terms of its subunit composition, optical and resonance Raman spectra, and cytochrome c oxidizing activity. When detergent extracts of spheroplasts from Paracoccus denitrificans were applied to these columns, the cytochrome c oxidase from this organism was also found to adhere tightly. Thus this purification method appears applicable to both prokaryotic and eukaryotic forms of the enzyme. The advantages of this new purification method are that it is less labor intensive than the traditional procedure and less expensive than methods based on cytochrome c-affinity chromatography.

  11. Specific affinity between fibronectin and the epidermolysis bullosa acquisita antigen.

    PubMed Central

    Woodley, D T; O'Keefe, E J; McDonald, J A; Reese, M J; Briggaman, R A; Gammon, W R

    1987-01-01

    Autoantibodies in the skin and sera of patients with epidermolysis bullosa acquisita bind to a large matrix molecule within the lamina densa region of skin basement membrane. At the site of these immune complexes, the epidermis separates from the dermis, which creates a subepidermal blister just below the lamina densa. The target molecule for the autoantibodies is in close apposition to fibronectin, a major extracellular matrix molecule that is abundant in the upper dermis of skin. In this report, we show specific affinity between fibronectin and the 290,000-D chain of the epidermolysis bullosa acquisita antigen, and that this affinity is mediated by the gelatin/collagen-binding domain of fibronectin (Mr = 60,000). Since blistering in epidermolysis bullosa acquisita often occurs in the absence of clinical and histological inflammation, a direct interruption in the fibronectin-epidermolysis bullosa acquisita antigen bond may be involved in the pathogenesis of epidermal-dermal disadherence that occurs in this bullous disease. Images PMID:3584471

  12. High-affinity carbamate analogues of morphinan at opioid receptors.

    PubMed

    Peng, Xuemei; Knapp, Brian I; Bidlack, Jean M; Neumeyer, John L

    2007-03-15

    A series of carbamate analogues were synthesized from levorphanol (1a), cyclorphan (2a) or butorphan (3a) and evaluated in vitro for their binding affinity at mu, delta, and kappa opioid receptors. Functional activities of these compounds were measured in the [(35)S]GTPgammaS binding assay. Phenyl carbamate derivatives 2d and 3d showed the highest binding affinity for kappa receptor (K(i)=0.046 and 0.051 nM) and for mu receptor (K(i)=0.11 and 0.12 nM). Compound 1c showed the highest mu selectivity. The preliminary assay for agonist and antagonist properties of these ligands in stimulating [(35)S]GTPgammaS binding mediated by the kappa opioid receptor illustrated that all of these ligands were kappa agonists. At the mu receptor, compounds 1b, 1c, 2b, and 3b were agonists, while compounds 2c-e and 3c-e were mu agonists/antagonists.

  13. Growth and mortality of larval Myctophum affine (Myctophidae, Teleostei).

    PubMed

    Namiki, C; Katsuragawa, M; Zani-Teixeira, M L

    2015-04-01

    The growth and mortality rates of Myctophum affine larvae were analysed based on samples collected during the austral summer and winter of 2002 from south-eastern Brazilian waters. The larvae ranged in size from 2·75 to 14·00 mm standard length (L(S)). Daily increment counts from 82 sagittal otoliths showed that the age of M. affine ranged from 2 to 28 days. Three models were applied to estimate the growth rate: linear regression, exponential model and Laird-Gompertz model. The exponential model best fitted the data, and L(0) values from exponential and Laird-Gompertz models were close to the smallest larva reported in the literature (c. 2·5 mm L(S)). The average growth rate (0·33 mm day(-1)) was intermediate among lanternfishes. The mortality rate (12%) during the larval period was below average compared with other marine fish species but similar to some epipelagic fishes that occur in the area.

  14. Flexible Molybdenum Electrodes towards Designing Affinity Based Protein Biosensors.

    PubMed

    Kamakoti, Vikramshankar; Panneer Selvam, Anjan; Radha Shanmugam, Nandhinee; Muthukumar, Sriram; Prasad, Shalini

    2016-07-18

    Molybdenum electrode based flexible biosensor on porous polyamide substrates has been fabricated and tested for its functionality as a protein affinity based biosensor. The biosensor performance was evaluated using a key cardiac biomarker; cardiac Troponin-I (cTnI). Molybdenum is a transition metal and demonstrates electrochemical behavior upon interaction with an electrolyte. We have leveraged this property of molybdenum for designing an affinity based biosensor using electrochemical impedance spectroscopy. We have evaluated the feasibility of detection of cTnI in phosphate-buffered saline (PBS) and human serum (HS) by measuring impedance changes over a frequency window from 100 mHz to 1 MHz. Increasing changes to the measured impedance was correlated to the increased dose of cTnI molecules binding to the cTnI antibody functionalized molybdenum surface. We achieved cTnI detection limit of 10 pg/mL in PBS and 1 ng/mL in HS medium. The use of flexible substrates for designing the biosensor demonstrates promise for integration with a large-scale batch manufacturing process.

  15. Iminobiotin affinity columns and their application to retrieval of streptavidin.

    PubMed Central

    Hofmann, K; Wood, S W; Brinton, C C; Montibeller, J A; Finn, F M

    1980-01-01

    A method is described for the retrieval of streptavidin from the culture broth of Streptomyces avidinii. The key step in this procedure is the adsorption of streptavidin from culture concentrates to an affinity column in which iminobiotin is attached to AH-Sepharose 4B. This column binds streptavbidin at pH 11 and releases the protein at pH 4. The recovery of streptavidin is practically quantitative. The pH dependence of the iminobiotin-avidin affinity, discovered by Green [Green, N. M. (1966) Biochem. J. 101, 774-779], has thus found practical application. The streptavidin bound 4.07 +/- 0.02 mol of [14C]biotin per mol and was essentially homogeneous as judged by disc and slab gel electrophoresis. Streptavidin was extensively succinoylated without loss of biotin-binding capacity. The observations that 125I-labeled streptavidin and 125I-labeled succinoylstreptavidin are retained by iminobiotin-AH-Sepharose 4B columns at pH 7.5 and are eluted at pH 4.0 provides a convenient purification method for these iodinated proteins. The technique employed for the retrieval of streptavidin is generally applicable to the isolation of iminobiotinylated molecules. PMID:6933515

  16. Membrane Affinity of Platensimycin and Its Dialkylamine Analogs

    PubMed Central

    Rowe, Ian; Guo, Min; Yasmann, Anthony; Cember, Abigail; Sintim, Herman O.; Sukharev, Sergei

    2015-01-01

    Membrane permeability is a desired property in drug design, but there have been difficulties in quantifying the direct drug partitioning into native membranes. Platensimycin (PL) is a new promising antibiotic whose biosynthetic production is costly. Six dialkylamine analogs of PL were synthesized with identical pharmacophores but different side chains; five of them were found inactive. To address the possibility that their activity is limited by the permeation step, we calculated polarity, measured surface activity and the ability to insert into the phospholipid monolayers. The partitioning of PL and the analogs into the cytoplasmic membrane of E. coli was assessed by activation curve shifts of a re-engineered mechanosensitive channel, MscS, in patch-clamp experiments. Despite predicted differences in polarity, the affinities to lipid monolayers and native membranes were comparable for most of the analogs. For PL and the di-myrtenyl analog QD-11, both carrying bulky sidechains, the affinity for the native membrane was lower than for monolayers (half-membranes), signifying that intercalation must overcome the lateral pressure of the bilayer. We conclude that the biological activity among the studied PL analogs is unlikely to be limited by their membrane permeability. We also discuss the capacity of endogenous tension-activated channels to detect asymmetric partitioning of exogenous substances into the native bacterial membrane and the different contributions to the thermodynamic force which drives permeation. PMID:26247942

  17. Electron attachment and detachment: Electron affinities of isomers of trifluoromethylbenzonitrile

    NASA Astrophysics Data System (ADS)

    Miller, Thomas M.; Viggiano, A. A.; Friedman, Jeffrey F.; Van Doren, Jane M.

    2004-11-01

    Rate constants for electron attachment to the three isomers of trifluoromethylbenzonitrile [(CF3)(CN)C6H4, or TFMBN] were measured over the temperature range of 303-463 K in a 133-Pa He buffer gas, using a flowing-afterglow Langmuir-probe apparatus. At 303 K, the measured attachment rate constants are 9.0×10-8 (o-TFMBN), 5.5×10-8 (m-TFMBN), and 8.9×10-8 cm3 s-1 (p-TFMBN), estimated accurate to ±25%. The attachment process formed only the parent anion in all three cases. Thermal electron detachment was observed for all three anion isomers, and rate constants for this reverse process were also measured. From the attachment and detachment results, the electron affinities of the three isomers of TFMBN were determined to be 0.70(o-TFMBN), 0.67(m-TFMBN), and 0.83 eV (p-TFMBN), all ±0.05 eV. G3(MP2) [Gaussian-3 calculations with reduced Møller-Plesset orders (MP2)] calculations were carried out for the neutrals and anions. Electron affinities derived from these calculations are in good agreement with the experimental values.

  18. Electron attachment and detachment: electron affinities of isomers of trifluoromethylbenzonitrile.

    PubMed

    Miller, Thomas M; Viggiano, A A; Friedman, Jeffrey F; Van Doren, Jane M

    2004-11-22

    Rate constants for electron attachment to the three isomers of trifluoromethylbenzonitrile [(CF(3))(CN)C(6)H(4), or TFMBN] were measured over the temperature range of 303-463 K in a 133-Pa He buffer gas, using a flowing-afterglow Langmuir-probe apparatus. At 303 K, the measured attachment rate constants are 9.0 x 10(-8) (o-TFMBN), 5.5 x 10(-8) (m-TFMBN), and 8.9 x 10(-8) cm(3) s(-1) (p-TFMBN), estimated accurate to +/-25%. The attachment process formed only the parent anion in all three cases. Thermal electron detachment was observed for all three anion isomers, and rate constants for this reverse process were also measured. From the attachment and detachment results, the electron affinities of the three isomers of TFMBN were determined to be 0.70(o-TFMBN), 0.67(m-TFMBN), and 0.83 eV (p-TFMBN), all +/-0.05 eV. G3(MP2) [Gaussian-3 calculations with reduced Møller-Plesset orders (MP2)] calculations were carried out for the neutrals and anions. Electron affinities derived from these calculations are in good agreement with the experimental values.

  19. Influence of affinity on antibody determination in microtiter ELISA systems

    SciTech Connect

    Peterman, J.H.; Voss, E.W. Jr.; Butler, J.E.

    1986-03-01

    Theoretically, all immunoassays are affinity (Ka) dependent when the product of the antibody (Ab) Ka and the free epitope concentration is less than 10. Thus, the degree of dependence on Ka depends on the concentration of available antigen in the system. The authors examined the binding of /sup 125/I-anti-fluorescein (a-FLU) monoclonal antibodies of different affinities to FLU-gelatin adsorbed on Immunlon 2 microtiter plates. Data obtained were in general agreement with our theoretical predictions; the percent of /sup 125/I-a-FLU which bound correlated with Ka, as did the shape of the titration curves. Measurement of 5 a-FLU monoclonals by the ELISA showed that the determination of Ab concentrations depends on the FLU-gelatin concentration, epitope density, and on the relationship between the Kas of test samples and the reference standard Ab preparation. Thus the ELISA is Ka dependent and should not be used routinely to estimate the absolute amount to Ab in unknown samples. However, the Ka dependency of the ELISA might provide a convenient assay for the estimation of the relative functional Ka (rfKa) of antibody preparations.

  20. Altered catecholamine receptor affinity in rabbit aortic intimal hyperplasia

    SciTech Connect

    O'Malley, M.K.; Cotecchia, S.; Hagen, P.O. )

    1991-08-01

    Intimal thickening is a universal response to endothelial denudation and is also thought to be a precursor of atherosclerosis. The authors have demonstrated selective supersensitivity in arterial intimal hyperplasia to norepinephrine and they now report a possible mechanism for this. Binding studies in rabbit aorta with the selective alpha 1-adrenergic radioligand 125I-HEAT demonstrated that there was no change in receptor density (20 {plus minus} 4 fmole/10(6) cells) in intact vascular smooth muscle cells at either 5 or 14 days after denudation. However, competition studies showed a 2.6-fold increase in alpha 1-adrenergic receptor affinity for norepinephrine in intimal hyperplastic tissue (P less than 0.05). This increased affinity for norepinephrine was associated with a greater increase in 32P-labeled phosphatidylinositol (148% intimal thickening versus 76% control) and phosphatidic acid (151% intimal thickening versus 56% control) following norepinephrine stimulation of free floating rings of intimal hyperplastic aorta. These data suggest that the catecholamine supersensitivity in rabbit aortic intimal hyperplasia is receptor mediated and may be linked to the phosphatidylinositol cycle.

  1. Binding Affinity of Glycoconjugates to BACILLUS Spores and Toxins

    NASA Astrophysics Data System (ADS)

    Rasol, Aveen; Eassa, Souzan; Tarasenko, Olga

    2010-04-01

    Early recognition of Bacillus cereus group species is important since they can cause food-borne illnesses and deadly diseases in humans. Glycoconjugates (GCs) are carbohydrates covalently linked to non-sugar moieties including lipids, proteins or other entities. GCs are involved in recognition and signaling processes intrinsic to biochemical functions in cells. They also stimulate cell-cell adhesion and subsequent recognition and activation of receptors. We have demonstrated that GCs are involved in Bacillus cereus spore recognition. In the present study, we have investigated whether GCs possess the ability to bind and recognize B. cereus spores and Bacillus anthracis recombinant single toxins (sTX) and complex toxins (cTX). The affinity of GCs to spores + sTX and spores + cTX toxins was studied in the binding essay. Our results demonstrated that GC9 and GC10 were able to selectively bind to B. cereus spores and B. anthracis toxins. Different binding affinities for GCs were found toward Bacillus cereus spores + sTX and spores + cTX. Dilution of GCs does not impede the recognition and binding. Developed method provides a tool for simultaneous recognition and targeting of spores, bacteria toxins, and/or other entities.

  2. Protein A affinity precipitation of human immunoglobulin G.

    PubMed

    Janoschek, Lars; Freiherr von Roman, Matthias; Berensmeier, Sonja

    2014-08-15

    The potential of protein A affinity precipitation as an alternative method for traditional antibody purification techniques was investigated. Recombinant produced protein A from Staphylococcus aureus (SpA) was covalently linked to the pH-responsive copolymer Eudragit(®) S-100 and used for purification of human immunoglobulin G (hIgG). The Eudragit-SpA conjugate had a static binding capacity of 93.9 ± 2.8 mg hIgG per g conjugate and a dissociation constant of 787 ± 67 nM at 7 ± 1°C. The antibody was adsorbed rapidly onto Eudragit-SpA and reached equilibrium within 5 min. An excess of hIgG binding sites, provided by the conjugate, as well as adjusted elution conditions resulted in an appropriate hIgG purification performance. In summary, Eudragit-SpA was successfully applied to capture hIgG from a protein mixture with 65% antibody yield in the elution step. Nearly 96% purity and a purification factor of 12.4 were achieved. The Eudragit-SpA conjugate showed a stable ligand density over several cycles, which enabled reusability for repeated precipitation of hIgG. According to this, pH induced affinity precipitation can be seen as a potential alternative for protein A chromatography in antibody purification processes.

  3. Benzodiazepines: electron affinity, receptors and cell signaling - a multifaceted approach.

    PubMed

    Kovacic, Peter; Ott, Nadia; Cooksy, Andrew L

    2013-12-01

    This report entails a multifaceted approach to benzodiazepine (BZ) action, involving electron affinity, receptors, cell signaling and other aspects. Computations of the electron affinities (EAs) of different BZs have been carried out to establish the effect of various substituents on their EA. These computations were undertaken to serve as a first step in determining what role electron transfer (ET) plays in BZ activity. The calculations were conducted on the premise that the nature of the substituent will either decrease or increase the electron density of the benzene ring, thus altering the ability of the molecule to accept an electron. Investigations were performed on the effect of drug protonation on EA. Similarities involving substituent effects in prior electrochemical studies are also discussed. As part of the multifaceted approach, EA is linked to ET, which appears to play a role in therapeutic activity and toxicity. There is extensive literature dealing with the role of receptors in BZ activity. Significant information on receptor involvement was reported more than 40 years ago. Gamma-aminobutyric acid (GABA) is known to be importantly involved. GABA is a probable mediator of BZ effects. BZ and GABA receptors, although not identical, are physiologically linked. Cell signaling is known to play a part in the biochemistry of BZ action. Various factors participated, such as gene expression, allosteric influence, toxic effects and therapeutic action. Evidence points to involvement of EA and ET in the mode of action in cell signaling. Oxidative stress and antioxidant effects are also addressed.

  4. Telonemia, a new protist phylum with affinity to chromist lineages

    PubMed Central

    Shalchian-Tabrizi, K; Eikrem, W; Klaveness, D; Vaulot, D; Minge, M.A; Le Gall, F; Romari, K; Throndsen, J; Botnen, A; Massana, R; Thomsen, H.A; Jakobsen, K.S

    2006-01-01

    Recent molecular investigations of marine samples taken from different environments, including tropical, temperate and polar areas, as well as deep thermal vents, have revealed an unexpectedly high diversity of protists, some of them forming deep-branching clades within important lineages, such as the alveolates and heterokonts. Using the same approach on coastal samples, we have identified a novel group of protist small subunit (SSU) rDNA sequences that do not correspond to any phylogenetic group previously identified. Comparison with other sequences obtained from cultures of heterotrophic protists showed that the environmental sequences grouped together with Telonema, a genus known since 1913 but of uncertain taxonomic affinity. Phylogenetic analyses using four genes (SSU, Hsp90, alpha-tubulin and beta-tubulin), and accounting for gamma- and covarion-distributed substitution rates, revealed Telonema as a distinct group of species branching off close to chromist lineages. Consistent with these gene trees, Telonema possesses ultrastructures revealing both the distinctness of the group and the evolutionary affinity to chromist groups. Altogether, the data suggest that Telonema constitutes a new eukaryotic phylum, here defined as Telonemia, possibly representing a key clade for the understanding of the early evolution of bikont protist groups, such as the proposed chromalveolate supergroup. PMID:16790418

  5. A global benchmark study using affinity-based biosensors

    PubMed Central

    Rich, Rebecca L.; Papalia, Giuseppe A.; Flynn, Peter J.; Furneisen, Jamie; Quinn, John; Klein, Joshua S.; Katsamba, Phini S.; Waddell, M. Brent; Scott, Michael; Thompson, Joshua; Berlier, Judie; Corry, Schuyler; Baltzinger, Mireille; Zeder-Lutz, Gabrielle; Schoenemann, Andreas; Clabbers, Anca; Wieckowski, Sebastien; Murphy, Mary M.; Page, Phillip; Ryan, Thomas E.; Duffner, Jay; Ganguly, Tanmoy; Corbin, John; Gautam, Satyen; Anderluh, Gregor; Bavdek, Andrej; Reichmann, Dana; Yadav, Satya P.; Hommema, Eric; Pol, Ewa; Drake, Andrew; Klakamp, Scott; Chapman, Trevor; Kernaghan, Dawn; Miller, Ken; Schuman, Jason; Lindquist, Kevin; Herlihy, Kara; Murphy, Michael B.; Bohnsack, Richard; Andrien, Bruce; Brandani, Pietro; Terwey, Danny; Millican, Rohn; Darling, Ryan J.; Wang, Liann; Carter, Quincy; Dotzlaf, Joe; Lopez-Sagaseta, Jacinto; Campbell, Islay; Torreri, Paola; Hoos, Sylviane; England, Patrick; Liu, Yang; Abdiche, Yasmina; Malashock, Daniel; Pinkerton, Alanna; Wong, Melanie; Lafer, Eileen; Hinck, Cynthia; Thompson, Kevin; Primo, Carmelo Di; Joyce, Alison; Brooks, Jonathan; Torta, Federico; Bagge Hagel, Anne Birgitte; Krarup, Janus; Pass, Jesper; Ferreira, Monica; Shikov, Sergei; Mikolajczyk, Malgorzata; Abe, Yuki; Barbato, Gaetano; Giannetti, Anthony M.; Krishnamoorthy, Ganeshram; Beusink, Bianca; Satpaev, Daulet; Tsang, Tiffany; Fang, Eric; Partridge, James; Brohawn, Stephen; Horn, James; Pritsch, Otto; Obal, Gonzalo; Nilapwar, Sanjay; Busby, Ben; Gutierrez-Sanchez, Gerardo; Gupta, Ruchira Das; Canepa, Sylvie; Witte, Krista; Nikolovska-Coleska, Zaneta; Cho, Yun Hee; D’Agata, Roberta; Schlick, Kristian; Calvert, Rosy; Munoz, Eva M.; Hernaiz, Maria Jose; Bravman, Tsafir; Dines, Monica; Yang, Min-Hsiang; Puskas, Agnes; Boni, Erica; Li, Jiejin; Wear, Martin; Grinberg, Asya; Baardsnes, Jason; Dolezal, Olan; Gainey, Melicia; Anderson, Henrik; Peng, Jinlin; Lewis, Mark; Spies, Peter; Trinh, Quyhn; Bibikov, Sergei; Raymond, Jill; Yousef, Mohammed; Chandrasekaran, Vidya; Feng, Yuguo; Emerick, Anne; Mundodo, Suparna; Guimaraes, Rejane; McGirr, Katy; Li, Yue-Ji; Hughes, Heather; Mantz, Hubert; Skrabana, Rostislav; Witmer, Mark; Ballard, Joshua; Martin, Loic; Skladal, Petr; Korza, George; Laird-Offringa, Ite; Lee, Charlene S.; Khadir, Abdelkrim; Podlaski, Frank; Neuner, Phillippe; Rothacker, Julie; Rafique, Ashique; Dankbar, Nico; Kainz, Peter; Gedig, Erk; Vuyisich, Momchilo; Boozer, Christina; Ly, Nguyen; Toews, Mark; Uren, Aykut; Kalyuzhniy, Oleksandr; Lewis, Kenneth; Chomey, Eugene; Pak, Brian J.; Myszka, David G.

    2013-01-01

    To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used. PMID:19133223

  6. Affinity Labeling of Membrane Receptors Using Tissue-Penetrating Radiations

    PubMed Central

    Wong, Franklin C.; Boja, John; Ho, Beng; Kuhar, Michael J.; Wong, Dean F.

    2013-01-01

    Photoaffinity labeling, a useful in vivo biochemical tool, is limited when applied in vivo because of the poor tissue penetration by ultraviolet (UV) photons. This study investigates affinity labeling using tissue-penetrating radiation to overcome the tissue attenuation and irreversibly label membrane receptor proteins. Using X-ray (115 kVp) at low doses (<50 cGy or Rad), specific and irreversible binding was found on striatal dopamine transporters with 3 photoaffinity ligands for dopamine transporters, to different extents. Upon X-ray exposure (115 kVp), RTI-38 and RTI-78 ligands showed irreversible and specific binding to the dopamine transporter similar to those seen with UV exposure under other conditions. Similarly, gamma rays at higher energy (662 keV) also affect irreversible binding of photoreactive ligands to peripheral benzodiazepine receptors (by PK14105) and to the dopamine (D2) membrane receptors (by azidoclebopride), respectively. This study reports that X-ray and gamma rays induced affinity labeling of membrane receptors in a manner similar to UV with photoreactive ligands of the dopamine transporter, D2 dopamine receptor (D2R), and peripheral benzodiazepine receptor (PBDZR). It may provide specific noninvasive irreversible block or stimulation of a receptor using tissue-penetrating radiation targeting selected anatomic sites. PMID:23936811

  7. Clinical Application of Ambient Ionization Mass Spectrometry

    PubMed Central

    Li, Li-Hua; Hsieh, Hua-Yi; Hsu, Cheng-Chih

    2017-01-01

    Ambient ionization allows mass spectrometry analysis directly on the sample surface under atmospheric pressure with almost zero sample pretreatment. Since the development of desorption electrospray ionization (DESI) in 2004, many other ambient ionization techniques were developed. Due to their simplicity and low operation cost, rapid and on-site clinical mass spectrometry analysis becomes real. In this review, we will highlight some of the most widely used ambient ionization mass spectrometry approaches and their applications in clinical study. PMID:28337399

  8. Quantifying protein interaction dynamics by SWATH mass spectrometry: application to the 14-3-3 system.

    PubMed

    Collins, Ben C; Gillet, Ludovic C; Rosenberger, George; Röst, Hannes L; Vichalkovski, Anton; Gstaiger, Matthias; Aebersold, Ruedi

    2013-12-01

    Protein complexes and protein interaction networks are essential mediators of most biological functions. Complexes supporting transient functions such as signal transduction processes are frequently subject to dynamic remodeling. Currently, the majority of studies on the composition of protein complexes are carried out by affinity purification and mass spectrometry (AP-MS) and present a static view of the system. For a better understanding of inherently dynamic biological processes, methods to reliably quantify temporal changes of protein interaction networks are essential. Here we used affinity purification combined with sequential window acquisition of all theoretical spectra (AP-SWATH) mass spectrometry to study the dynamics of the 14-3-3β scaffold protein interactome after stimulation of the insulin-PI3K-AKT pathway. The consistent and reproducible quantification of 1,967 proteins across all stimulation time points provided insights into the 14-3-3β interactome and its dynamic changes following IGF1 stimulation. We therefore establish AP-SWATH as a tool to quantify dynamic changes in protein-complex interaction networks.

  9. A mass spectrometry primer for mass spectrometry imaging

    PubMed Central

    Rubakhin, Stanislav S.; Sweedler, Jonathan V.

    2011-01-01

    Mass spectrometry imaging (MSI), a rapidly growing subfield of chemical imaging, employs mass spectrometry (MS) technologies to create single- and multi-dimensional localization maps for a variety of atoms and molecules. Complimentary to other imaging approaches, MSI provides high chemical specificity and broad analyte coverage. This powerful analytical toolset is capable of measuring the distribution of many classes of inorganics, metabolites, proteins and pharmaceuticals in chemically and structurally complex biological specimens in vivo, in vitro, and in situ. The MSI approaches highlighted in this Methods in Molecular Biology volume provide flexibility of detection, characterization, and identification of multiple known and unknown analytes. The goal of this chapter is to introduce investigators who may be unfamiliar with MS to the basic principles of the mass spectrometric approaches as used in MSI. In addition to guidelines for choosing the most suitable MSI method for specific investigations, cross-references are provided to the chapters in this volume that describe the appropriate experimental protocols. PMID:20680583

  10. Mass spectrometry of aerospace materials

    NASA Technical Reports Server (NTRS)

    Colony, J. A.

    1976-01-01

    Mass spectrometry is used for chemical analysis of aerospace materials and contaminants. Years of analytical aerospace experience have resulted in the development of specialized techniques of sampling and analysis which are required in order to optimize results. This work has resulted in the evolution of a hybrid method of indexing mass spectra which include both the largest peaks and the structurally significant peaks in a concise format. With this system, a library of mass spectra of aerospace materials was assembled, including the materials responsible for 80 to 90 percent of the contamination problems at Goddard Space Flight Center during the past several years.

  11. Mass spectrometry. [review of techniques

    NASA Technical Reports Server (NTRS)

    Burlingame, A. L.; Kimble, B. J.; Derrick, P. J.

    1976-01-01

    Advances in mass spectrometry (MS) and its applications over the past decade are reviewed in depth, with annotated literature references. New instrumentation and techniques surveyed include: modulated-beam MS, chromatographic MS on-line computer techniques, digital computer-compatible quadrupole MS, selected ion monitoring (mass fragmentography), and computer-aided management of MS data and interpretation. Areas of application surveyed include: organic MS and electron impact MS, field ionization kinetics, appearance potentials, translational energy release, studies of metastable species, photoionization, calculations of molecular orbitals, chemical kinetics, field desorption MS, high pressure MS, ion cyclotron resonance, biochemistry, medical/clinical chemistry, pharmacology, and environmental chemistry and pollution studies.

  12. High Technology Mass Spectrometry Laboratory

    DTIC Science & Technology

    2010-08-01

    GSH, hemoglobin beta-Cys93 ( Hb -C93-AN) were monitored. The second order rate constants in M-ls-1 were: disappe 0.0806; appearance of GS-AN in whole...blood, 0.0776, appearance of Hb -C9 appearance of AbC34-AN in plasma, 0.224. The data indicate that the mos blood is Cys34 of albumin. This site...than Hb -C93 15. SUBJECT TERMS acrylonitrile, adduct, mass spectrometry, biomarker, toxic industrial chemicals 16. SECURITY CLASSIFICATION OF: a

  13. Glycosaminoglycan Glycomics Using Mass Spectrometry*

    PubMed Central

    Zaia, Joseph

    2013-01-01

    The fact that sulfated glycosaminoglycans (GAGs) are necessary for the functioning of all animal physiological systems drives the need to understand their biology. This understanding is limited, however, by the heterogeneous nature of GAG chains and their dynamic spatial and temporal expression patterns. GAGs have a regulated structure overlaid by heterogeneity but lack the detail necessary to build structure/function relationships. In order to provide this information, we need glycomics platforms that are sensitive, robust, high throughput, and information rich. This review summarizes progress on mass-spectrometry-based GAG glycomics methods. The areas covered include disaccharide analysis, oligosaccharide profiling, and tandem mass spectrometric sequencing. PMID:23325770

  14. Familial secondary erythrocytosis due to increased oxygen affinity is caused by destabilization of the T state of hemoglobin Brigham (α2β2Pro100Leu)

    PubMed Central

    Mollan, Todd L; Abraham, Bindu; Strader, Michael Brad; Jia, Yiping; Lozier, Jay N; Olson, John S; Alayash, Abdu I

    2012-01-01

    Hemoglobin Brigham (β Pro100 to Leu) was first reported in a patient with familial erythrocytosis. Erythrocytes of an affected individual from the same family contain both HbA and Hb Brigham and exhibit elevated O2 affinity compared with normal cells (P50 = 23 mm Hg vs. 31 mmHg at pH 7.4 at 37°C). O2 affinities measured for hemolysates were sensitive to changes in pH or chloride concentrations, indicating little change in the Bohr and Chloride effects. Hb Brigham was separated from normal HbA by nondenaturing cation exchange liquid chromatography, and the amino acid substitution was verified by mass spectrometry. The properties of Hb Brigham isolated from the patient's blood were then compared with those of recombinant Hb Brigham expressed in Escherichia coli. Kinetic experiments suggest that the rate constants for ligand binding and release in the high (R) and low (T) affinity quaternary states of Hb Brigham are similar to those of native hemoglobin. However, the Brigham mutation decreases the T to R equilibrium constant (L) which accelerates the switch to the R state during ligand binding to deoxy-Hb, increasing the rate of association by approximately twofold, and decelerates the switch during ligand dissociation from HbO2, decreasing the rate approximately twofold. These kinetic data help explain the high O2 affinity characteristics of Hb Brigham and provide further evidence for the importance of the contribution of Pro100 to intersubunit contacts and stabilization of the T quaternary structure. PMID:22821886

  15. Triple bioaffinity mass spectrometry concept for thyroid transporter ligands.

    PubMed

    Aqai, Payam; Fryganas, Christos; Mizuguchi, Mineyuki; Haasnoot, Willem; Nielen, Michel W F

    2012-08-07

    For the analysis of thyroid transporter ligands, a triple bioaffinity mass spectrometry (BioMS) concept was developed, with the aim at three different analytical objectives: rapid screening of any ligand, confirmation of known ligands in accordance with legislative requirements, and identification of emerging yet unknown ligands. These three purposes share the same biorecognition element, recombinant thyroid transport protein transthyretin (rTTR), and dedicated modes of liquid chromatography-mass spectrometry (LC-MS). For screening, a rapid and radiolabel-free competitive inhibition MS binding assay was developed with fast ultrahigh performance-liquid chromatography-electrospray ionization-triple-quadrupole-MS (UPLC-QqQ-MS) as the readout system. It uses the nonradioactive stable isotopic thyroid hormone (13)C(6)-L-thyroxine as the label of which the binding to rTTR is inhibited by any ligand such as thyroid drugs and thyroid endocrine disrupting chemicals (EDCs). To this end, rTTR is either used in solution or immobilized on paramagnetic microbeads. The concentration-dependent inhibition of the label by the natural thyroid hormone l-thyroxine (T4), as a model analyte, is demonstrated in water at part-per-trillion and in urine at part-per-billion level. For confirmation of identity of known ligands, rTTR was used for bioaffinity purification for confirmation of naturally present free T4 in urine. As a demonstrator for identification of unknown ligands, the same rTTR was used again but in combination with nano-UPLC-quadrupole time-of-flight-MS (nLC-Q-TOF-MS) and urine samples spiked with the model "unknown" EDCs triclosan and tetrabromobisphenol-A. This study highlights the potential of BioMS using one affinity system, both for rapid screening and for confirmation and identification of known and unknown emerging thyroid EDCs.

  16. Low-affinity Na+ uptake in the halophyte Suaeda maritima.

    PubMed

    Wang, Suo-Min; Zhang, Jin-Lin; Flowers, Timothy J

    2007-10-01

    Na(+) uptake by plant roots has largely been explored using species that accumulate little Na(+) into their shoots. By way of contrast, the halophyte Suaeda maritima accumulates, without injury, concentrations of the order of 400 mM NaCl in its leaves. Here we report that cAMP and Ca(2+) (blockers of nonselective cation channels) and Li(+) (a competitive inhibitor of Na(+) uptake) did not have any significant effect on the uptake of Na(+) by the halophyte S. maritima when plants were in 25 or 150 mM NaCl (150 mM NaCl is near optimal for growth). However, the inhibitors of K(+) channels, TEA(+) (10 mM), Cs(+) (3 mM), and Ba(2+) (5 mM), significantly reduced the net uptake of Na(+) from 150 mM NaCl over 48 h, by 54%, 24%, and 29%, respectively. TEA(+) (10 mM), Cs(+) (3 mM), and Ba(2+) (1 mm) also significantly reduced (22)Na(+) influx (measured over 2 min in 150 mM external NaCl) by 47%, 30%, and 31%, respectively. In contrast to the situation in 150 mm NaCl, neither TEA(+) (1-10 mM) nor Cs(+) (0.5-10 mM) significantly reduced net Na(+) uptake or (22)Na(+) influx in 25 mM NaCl. Ba(2+) (at 5 mm) did significantly decrease net Na(+) uptake (by 47%) and (22)Na(+) influx (by 36% with 1 mM Ba(2+)) in 25 mM NaCl. K(+) (10 or 50 mM) had no effect on (22)Na(+) influx at concentrations below 75 mM NaCl, but the influx of (22)Na(+) was inhibited by 50 mM K(+) when the external concentration of NaCl was above 75 mM. The data suggest that neither nonselective cation channels nor a low-affinity cation transporter are major pathways for Na(+) entry into root cells. We propose that two distinct low-affinity Na(+) uptake pathways exist in S. maritima: Pathway 1 is insensitive to TEA(+) or Cs(+), but sensitive to Ba(2+) and mediates Na(+) uptake under low salinities (25 mM NaCl); pathway 2 is sensitive to TEA(+), Cs(+), and Ba(2+) and mediates Na(+) uptake under higher external salt concentrations (150 mM NaCl). Pathway 1 might be mediated by a high-affinity K transporter

  17. Picomolar affinity fibronectin domains engineered utilizing loop length diversity, recursive mutagenesis, and loop shuffling.

    PubMed

    Hackel, Benjamin J; Kapila, Atul; Wittrup, K Dane

    2008-09-19

    The 10th type III domain of human fibronectin (Fn3) has been validated as an effective scaffold for molecular recognition. In the current work, it was desired to improve the robustness of selection of stable, high-affinity Fn3 domains. A yeast surface display library of Fn3 was created in which three solvent-exposed loops were diversified in terms of amino acid composition and loop length. The library was screened by fluorescence-activated cell sorting to isolate binders to lysozyme. An affinity maturation scheme was developed to rapidly and broadly diversify populations of clones by random mutagenesis as well as homologous recombination-driven shuffling of mutagenized loops. The novel library and affinity maturation scheme combined to yield stable, monomeric Fn3 domains with 3 pM affinity for lysozyme. A secondary affinity maturation identified a stable 1.1 pM binder, the highest affinity yet reported for an Fn3 domain. In addition to extension of the affinity limit for this scaffold, the results demonstrate the ability to achieve high-affinity binding while preserving stability and the monomeric state. This library design and affinity maturation scheme is highly efficient, utilizing an initial diversity of 2x10(7) clones and screening only 1x10(8) mutants (totaled over all affinity maturation libraries). Analysis of intermediate populations revealed that loop length diversity, loop shuffling, and recursive mutagenesis of diverse populations are all critical components.

  18. Interaction of natural polyphenols with α-amylase in vitro: molecular property-affinity relationship aspect.

    PubMed

    Xiao, Jianbo; Kai, Guoyin; Ni, Xiaoling; Yang, Fan; Chen, Xiaoqing

    2011-06-01

    The relationship between the structural properties of natural polyphenols and their affinities for α-amylase were investigated by fluorescence titration analysis. The binding process with α-amylase was strongly influenced by the structural differences of the compounds under study. For instance, the methylation of the hydroxyl group in flavonoids increased their binding affinities for α-amylase by 2.14 to 7.76 times. The hydroxylation on rings A, B, and C of flavonoids also significantly affected their affinities for α-amylase. The glycosylation of isoflavones and flavanones reduced their affinities for α-amylase and the glycosylation of flavones and flavonols enhanced their affinities for α-amylase. Hydrogenation of the C2=C3 double bond of flavonoids decreased the binding affinities. The galloylated catechins had higher binding affinities with α-amylase than non-galloylated catechins and the pyrogallol-type catechins had higher affinities than the catechol-type catechins. The presence of the galloyl moiety is the most decisive factor. The glycosylation of resveratrol decreased its affinity for α-amylase. The esterification of gallic acid significantly reduced the affinity for α-amylase. The binding interaction between polyphenols and α-amylase was mainly caused by hydrophobic forces.

  19. Quantitative mass spectrometry: an overview

    NASA Astrophysics Data System (ADS)

    Urban, Pawel L.

    2016-10-01

    Mass spectrometry (MS) is a mainstream chemical analysis technique in the twenty-first century. It has contributed to numerous discoveries in chemistry, physics and biochemistry. Hundreds of research laboratories scattered all over the world use MS every day to investigate fundamental phenomena on the molecular level. MS is also widely used by industry-especially in drug discovery, quality control and food safety protocols. In some cases, mass spectrometers are indispensable and irreplaceable by any other metrological tools. The uniqueness of MS is due to the fact that it enables direct identification of molecules based on the mass-to-charge ratios as well as fragmentation patterns. Thus, for several decades now, MS has been used in qualitative chemical analysis. To address the pressing need for quantitative molecular measurements, a number of laboratories focused on technological and methodological improvements that could render MS a fully quantitative metrological platform. In this theme issue, the experts working for some of those laboratories share their knowledge and enthusiasm about quantitative MS. I hope this theme issue will benefit readers, and foster fundamental and applied research based on quantitative MS measurements. This article is part of the themed issue 'Quantitative mass spectrometry'.

  20. Mass spectrometry in combinatorial chemistry.

    PubMed

    Enjalbal, C; Martinez, J; Aubagnac, J L

    2000-01-01

    In the fast expanding field of combinatorial chemistry, profiling libraries has always been a matter of concern--as illustrated by the buoyant literature over the past seven years. Spectroscopic methods, including especially mass spectrometry and to a lesser extent IR and NMR, have been applied at different levels of combinatorial library synthesis: in the rehearsal phase to optimize the chemistry prior to library generation, to confirm library composition, and to characterize after screening each structure that exhibits positive response. Most of the efforts have been concentrated on library composition assessment. The difficulties of such analyses have evolved from the infancy of the combinatorial concept, where large mixtures were prepared, to the recent parallel syntheses of collections of discrete compounds. Whereas the complexity of the analyses has diminished, an increased degree of automation was simultaneously required to achieve efficient library component identification and quantification. In this respect, mass spectrometry has been found to be the method of choice, providing rapid, sensitive, and informative analyses, especially when coupled to chromatographic separation. Fully automated workstations able to cope with several hundreds of compounds per day have been designed. After a brief introduction to describe the combinatorial approach, library characterization will be discussed in detail, considering first the solution-based methodologies and secondly the support-bound material analyses.

  1. Mass Spectrometry Applications for Toxicology

    PubMed Central

    Mbughuni, Michael M.; Jannetto, Paul J.

    2016-01-01

    Toxicology is a multidisciplinary study of poisons, aimed to correlate the quantitative and qualitative relationships between poisons and their physiological and behavioural effects in living systems. Other key aspects of toxicology focus on elucidation of the mechanisms of action of poisons and development of remedies and treatment plans for associated toxic effects. In these endeavours, Mass spectrometry (MS) has become a powerful analytical technique with a wide range of application used in the Toxicological analysis of drugs, poisons, and metabolites of both. To date, MS applications have permeated all fields of toxicology which include; environmental, clinical, and forensic toxicology. While many different analytical applications are used in these fields, MS and its hyphenated applications such as; gas chromatography MS (GC-MS), liquid chromatography MS (LC-MS), inductively coupled plasma ionization MS (ICP-MS), tandem mass spectrometry (MS/MS and MSn) have emerged as powerful tools used in toxicology laboratories. This review will focus on these hyphenated MS technologies and their applications for toxicology. PMID:28149262

  2. Mass Spectrometry Applications for Toxicology.

    PubMed

    Mbughuni, Michael M; Jannetto, Paul J; Langman, Loralie J

    2016-12-01

    Toxicology is a multidisciplinary study of poisons, aimed to correlate the quantitative and qualitative relationships between poisons and their physiological and behavioural effects in living systems. Other key aspects of toxicology focus on elucidation of the mechanisms of action of poisons and development of remedies and treatment plans for associated toxic effects. In these endeavours, Mass spectrometry (MS) has become a powerful analytical technique with a wide range of application used in the Toxicological analysis of drugs, poisons, and metabolites of both. To date, MS applications have permeated all fields of toxicology which include; environmental, clinical, and forensic toxicology. While many different analytical applications are used in these fields, MS and its hyphenated applications such as; gas chromatography MS (GC-MS), liquid chromatography MS (LC-MS), inductively coupled plasma ionization MS (ICP-MS), tandem mass spectrometry (MS/MS and MS(n)) have emerged as powerful tools used in toxicology laboratories. This review will focus on these hyphenated MS technologies and their applications for toxicology.

  3. Atmospheric pressure ionization-tandem mass spectrometry of the phenicol drug family.

    PubMed

    Alechaga, Élida; Moyano, Encarnación; Galceran, M Teresa

    2013-11-01

    In this work, the mass spectrometry behaviour of the veterinary drug family of phenicols, including chloramphenicol (CAP) and its related compounds thiamphenicol (TAP), florfenicol (FF) and FF amine (FFA), was studied. Several atmospheric pressure ionization sources, electrospray (ESI), atmospheric pressure chemical ionization and atmospheric pressure photoionization were compared. In all atmospheric pressure ionization sources, CAP, TAP and FF were ionized in both positive and negative modes; while for the metabolite FFA, only positive ionization was possible. In general, in positive mode, [M + H](+) dominated the mass spectrum for FFA, while the other compounds, CAP, TAP and FF, with lower proton affinity showed intense adducts with species present in the mobile phase. In negative mode, ESI and atmospheric pressure photoionization showed the deprotonated molecule [M-H](-), while atmospheric pressure chemical ionization provided the radical molecular ion by electron capture. All these ions were characterized by tandem mass spectrometry using the combined information obtained by multistage mass spectrometry and high-resolution mass spectrometry in a quadrupole-Orbitrap instrument. In general, the fragmentation occurred via cyclization and losses or fragmentation of the N-(alkyl)acetamide group, and common fragmentation pathways were established for this family of compounds. A new chemical structure for the product ion at m/z 257 for CAP, on the basis of the MS(3) and MS(4) spectra is proposed. Thermally assisted ESI and selected reaction monitoring are proposed for the determination of these compounds by ultra high-performance liquid chromatography coupled to tandem mass spectrometry, achieving instrumental detection limits down to 0.1 pg.

  4. Application of a New Dual Localization-Affinity Purification Tag Reveals Novel Aspects of Protein Kinase Biology in Aspergillus nidulans

    PubMed Central

    De Souza, Colin P.; Hashmi, Shahr B.; Osmani, Aysha H.; Osmani, Stephen A.

    2014-01-01

    Filamentous fungi occupy critical environmental niches and have numerous beneficial industrial applications but devastating effects as pathogens and agents of food spoilage. As regulators of essentially all biological processes protein kinases have been intensively studied but how they regulate the often unique biology of filamentous fungi is not completely understood. Significant understanding of filamentous fungal biology has come from the study of the model organism Aspergillus nidulans using a combination of molecular genetics, biochemistry, cell biology and genomic approaches. Here we describe dual localization-affinity purification (DLAP) tags enabling endogenous N or C-terminal protein tagging for localization and biochemical studies in A. nidulans. To establish DLAP tag utility we endogenously tagged 17 protein kinases for analysis by live cell imaging and affinity purification. Proteomic analysis of purifications by mass spectrometry confirmed association of the CotA and NimXCdk1 kinases with known binding partners and verified a predicted interaction of the SldABub1/R1 spindle assembly checkpoint kinase with SldBBub3. We demonstrate that the single TOR kinase of A. nidulans locates to vacuoles and vesicles, suggesting that the function of endomembranes as major TOR cellular hubs is conserved in filamentous fungi. Comparative analysis revealed 7 kinases with mitotic specific locations including An-Cdc7 which unexpectedly located to mitotic spindle pole bodies (SPBs), the first such localization described for this family of DNA replication kinases. We show that the SepH septation kinase locates to SPBs specifically in the basal region of apical cells in a biphasic manner during mitosis and again during septation. This results in gradients of SepH between G1 SPBs which shift along hyphae as each septum forms. We propose that SepH regulates the septation initiation network (SIN) specifically at SPBs in the basal region of G1 cells and that localized gradients

  5. Fully activated MEK1 exhibits compromised affinity for binding of allosteric inhibitors U0126 and PD0325901.

    PubMed

    Sheth, Payal R; Liu, Yuqi; Hesson, Thomas; Zhao, Jia; Vilenchik, Lev; Liu, Yan-Hui; Mayhood, Todd W; Le, Hung V

    2011-09-20

    Kinases catalyze the transfer of γ-phosphate from ATP to substrate protein residues triggering signaling pathways responsible for a plethora of cellular events. Isolation and production of homogeneous preparations of kinases in their fully active forms is important for accurate in vitro measurements of activity, stability, and ligand binding properties of these proteins. Previous studies have shown that MEK1 can be produced in its active phosphorylated form by coexpression with RAF1 in insect cells. In this study, using activated MEK1 produced by in vitro activation by RAF1 (pMEK1(in vitro)), we demonstrate that the simultaneous expression of RAF1 for production of activated MEK1 does not result in stoichiometric phosphorylation of MEK1. The pMEK1(in vitro) showed higher specific activity toward ERK2 protein substrate compared to the pMEK1 that was activated via coexpression with RAF1 (pMEK1(in situ)). The two pMEK1 preparations showed quantitative differences in the phosphorylation of T-loop residue serine 222 by Western blotting and mass spectrometry. Finally, pMEK1(in vitro) showed marked differences in the ligand binding properties compared to pMEK1(in situ). Contrary to previous findings, pMEK1(in vitro) bound allosteric inhibitors U0126 and PD0325901 with a significantly lower affinity than pMEK1(in situ) as well as its unphosphorylated counterpart (npMEK1) as demonstrated by thermal-shift, AS-MS, and calorimetric studies. The differences in inhibitor binding affinity provide direct evidence that unphosphorylated and RAF1-phosphorylated MEK1 form distinct inhibitor sites.

  6. An Affinity Propagation-Based DNA Motif Discovery Algorithm.

    PubMed

    Sun, Chunxiao; Huo, Hongwei; Yu, Qiang; Guo, Haitao; Sun, Zhigang

    2015-01-01

    The planted (l, d) motif search (PMS) is one of the fundamental problems in bioinformatics, which plays an important role in locating transcription factor binding sites (TFBSs) in DNA sequences. Nowadays, identifying weak motifs and reducing the effect of local optimum are still important but challenging tasks for motif discovery. To solve the tasks, we propose a new algorithm, APMotif, which first applies the Affinity Propagation (AP) clustering in DNA sequences to produce informative and good candidate motifs and then employs Expectation Maximization (EM) refinement to obtain the optimal motifs from the candidate motifs. Experimental results both on simulated data sets and real biological data sets show that APMotif usually outperforms four other widely used algorithms in terms of high prediction accuracy.

  7. Affinity sensor based on immobilized molecular imprinted synthetic recognition elements.

    PubMed

    Lenain, Pieterjan; De Saeger, Sarah; Mattiasson, Bo; Hedström, Martin

    2015-07-15

    An affinity sensor based on capacitive transduction was developed to detect a model compound, metergoline, in a continuous flow system. This system simulates the monitoring of low-molecular weight organic compounds in natural flowing waters, i.e. rivers and streams. During operation in such scenarios, control of the experimental parameters is not possible, which poses a true analytical challenge. A two-step approach was used to produce a sensor for metergoline. Submicron spherical molecularly imprinted polymers, used as recognition elements, were obtained through emulsion polymerization and subsequently coupled to the sensor surface by electropolymerization. This way, a robust and reusable sensor was obtained that regenerated spontaneously under the natural conditions in a river. Small organic compounds could be analyzed in water without manipulating the binding or regeneration conditions, thereby offering a viable tool for on-site application.

  8. High-affinity ammonium transporters and nitrogen sensing in mycorrhizas.

    PubMed

    Javelle, Arnaud; André, Bruno; Marini, Anne Marie; Chalot, Michel

    2003-02-01

    Most terrestrial plants live in mutualistic symbiosis with root-infecting mycorrhizal fungi. This association requires a molecular dialogue between the two partners. However, the nature of the chemical signals that induce hyphal differentiation are not well characterized and the mechanisms for signal reception are still unknown. In addition to its role in ammonium scavenging, the Mep2 protein from Saccharomyces cerevisiae has been proposed to act as an ammonium sensor that is essential for pseudohyphal differentiation in response to ammonium limitation. We propose that the high-affinity ammonium transporters from mycorrhizal fungi act in a similar manner to sense the environment and induce, via as-yet-unidentified signal transduction cascades, the switch in the mode of fungal growth observed during the formation of mycorrhiza.

  9. Predicting protein-ligand affinity with a random matrix framework.

    PubMed

    Lee, Alpha A; Brenner, Michael P; Colwell, Lucy J

    2016-11-29

    Rapid determination of whether a candidate compound will bind to a particular target receptor remains a stumbling block in drug discovery. We use an approach inspired by random matrix theory to decompose the known ligand set of a target in terms of orthogonal "signals" of salient chemical features, and distinguish these from the much larger set of ligand chemical features that are not relevant for binding to that particular target receptor. After removing the noise caused by finite sampling, we show that the similarity of an unknown ligand to the remaining, cleaned chemical features is a robust predictor of ligand-target affinity, performing as well or better than any algorithm in the published literature. We interpret our algorithm as deriving a model for the binding energy between a target receptor and the set of known ligands, where the underlying binding energy model is related to the classic Ising model in statistical physics.

  10. Chelators whose affinity for calcium is decreased by illumination

    NASA Technical Reports Server (NTRS)

    Tsien, Roger Y. (Inventor); Grynkiewicz, Grzegorz (Inventor); Minta, Akwasi (Inventor)

    1987-01-01

    The present invention discloses a group of calcium chelating compounds which have a descreased affinity for calcium following illumination. These new compounds contain a photolabile nitrobenzyl derivative coupled to a tetracarboxylate Ca.sup.2+ chelating parent compound having the octacoordinate chelating groups characteristic of EGTA or BAPTA. In a first form, the new compounds are comprised of a BAPTA-like chelator coupled to a single 2-nitrobenzyl derivative, which in turn is a photochemical precursor of a 2-nitrosobenzophenone. In a second form, the new compounds are comprised of a BAPTA-like chelator coupled to two 2-nitrobenzyl derivatives, themselves photochemical prcursors of the related 2-nitrosobenzophenones. The present invention also discloses a novel method for preparing 1-hydroxy- or 1-alkoxy-1-(2-nitroaryl)-1-aryl methanes. Methanes of this type are critical to the preparation of, or actually constitute, the photolabile Ca.sup.2+ chelating compounds disclosed and claimed herein.

  11. Bioavailable affinity label for collagen prolyl 4-hydroxylase

    PubMed Central

    Vasta, James D.; Higgin, Joshua J.; Kersteen, Elizabeth A.

    2013-01-01

    Collagen is the most abundant protein in animals. Its prevalent 4-hydroxyproline residues contribute greatly to its conformational stability. The hydroxyl groups arise from a post-translational modification catalyzed by the non-heme iron-dependent enzyme, collagen prolyl 4-hydroxylase (P4H). Here, we report that 4-oxo-5,6-epoxyhexanoate, a mimic of the α-ketoglutarate co-substrate, inactivates human P4H. The inactivation installs a ketone functionality in P4H, providing a handle for proteomic experiments. Caenorhabditis elegans exposed to the esterified epoxy ketone displays the phenotype of a worm lacking P4H. Thus, this affinity label can be used to mediate collagen stability in an animal, as is desirable in the treatment of a variety of fibrotic diseases. PMID:23702396

  12. HIGH AFFINITY, DSRNA BINDING BY DISCONNECTED INTERACTING PROTEIN 1†

    PubMed Central

    Catanese, Daniel J.; Matthews, Kathleen S.

    2010-01-01

    Disconnected Interacting Protein 1 (DIP1) appears from sequence analysis and preliminary binding studies to be a member of the dsRNA-binding protein family. Of interest, DIP1 was shown previously to interact with and influence multiple proteins involved in transcription regulation in Drosophila melanogaster. We show here that the longest isoform of this protein, DIP1-c, exhibits a 500-fold preference for dsRNA over dsDNA of similar nucleotide sequence. Further, DIP1-c demonstrated very high affinity for a subset of dsRNA ligands, with binding in the picomolar range for VA1 RNA and miR-iab-4 precursor stem-loop, a potential physiological RNA target involved in regulating expression of its protein partner, Ultrabithorax. PMID:20643095

  13. Approximated affine projection algorithm for feedback cancellation in hearing aids.

    PubMed

    Lee, Sangmin; Kim, In-Young; Park, Young-Cheol

    2007-09-01

    We propose an approximated affine projection (AP) algorithm for feedback cancellation in hearing aids. It is based on the conventional approach using the Gauss-Seidel (GS) iteration, but provides more stable convergence behaviour even with small step sizes. In the proposed algorithm, a residue of the weighted error vector, instead of the current error sample, is used to provide stable convergence. A new learning rate control scheme is also applied to the proposed algorithm to prevent signal cancellation and system instability. The new scheme determines step size in proportion to the prediction factor of the input, so that adaptation is inhibited whenever tone-like signals are present in the input. Simulation results verified the efficiency of the proposed algorithm.

  14. Genetic affinities of Ukrainians from the maternal perspective.

    PubMed

    Pshenichnov, Andrey; Balanovsky, Oleg; Utevska, Olga; Metspalu, Ene; Zaporozhchenko, Valery; Agdzhoyan, Anastasia; Churnosov, Mikhail; Atramentova, Lyubov; Balanovska, Elena

    2013-12-01

    The area of what is now the Ukraine has been the arena of large-scale demographic processes that may have left their traces in the contemporary gene pool of Ukrainians. In this study, we present new mitochondrial DNA data for 607 Ukrainians (hypervariable segment I sequences and coding region polymorphisms). To study the maternal affinities of Ukrainians at the level of separate mitochondrial haplotypes, we apply an original technique, the haplotype co-occurrence analysis. About 20% of the Ukrainian maternal gene pool is represented by lineages highly specific to Ukrainians, but is scarcely found in other populations. About 9% of Ukrainian mtDNA lineages are typical for peoples of the Volga region. We also identified minor gene pool strata (1.6-3.3%), each of which is common in Lithuanians, Estonians, Saami, Nenets, Cornish, and the populations of the North Caucasus.

  15. Deformation of supersymmetric and conformal quantum mechanics through affine transformations

    NASA Technical Reports Server (NTRS)

    Spiridonov, Vyacheslav

    1993-01-01

    Affine transformations (dilatations and translations) are used to define a deformation of one-dimensional N = 2 supersymmetric quantum mechanics. Resulting physical systems do not have conserved charges and degeneracies in the spectra. Instead, superpartner Hamiltonians are q-isospectral, i.e. the spectrum of one can be obtained from another (with possible exception of the lowest level) by q(sup 2)-factor scaling. This construction allows easily to rederive a special self-similar potential found by Shabat and to show that for the latter a q-deformed harmonic oscillator algebra of Biedenharn and Macfarlane serves as the spectrum generating algebra. A general class of potentials related to the quantum conformal algebra su(sub q)(1,1) is described. Further possibilities for q-deformation of known solvable potentials are outlined.

  16. Calculation of Electron Affinity and Partial Cross Sections of Hf^-

    NASA Astrophysics Data System (ADS)

    Pan, Lin; Beck, Donald

    2008-05-01

    We have calculated for the first time the electron affinity (EA) of Hf^-, using the relativistic configuration interaction method. Our calculations show Hf^- has only one bound state 5d^26s^26p J=5/2, which is a 6p attachment to the ground state of Hf I. By combining our valence stage result with the separate estimate for the modest core-valence contribution, the EA of Hf^- is about 0.114 eV. So far there have been only two experimental results [1,2] for the EA of Hf^-, but both gave only the limits. Our result falls within both of the limits. We also calculate the partial cross sections for photodetachment to the lower lying neutral thresholds. [1] M-J. Nadeau et al, Nucl. Instr. and Meth. B 123, 521 (1997) [2] Vernon T. Davis et al, Nucl. Instr. and Meth. B 241, 118 (2005)

  17. Self-affine crossover length in a layered silicate deposit.

    PubMed

    Fossum, J O; Bergene, H H; Hansen, Alex; O'Rourke, B; Manificat, G

    2004-03-01

    Self-affine dehydrated colloidal deposits on fresh mica surfaces of the synthetic layered silicate 2:1 smectite clay laponite have been studied by means of atomic force microscopy (AFM). AFM images of these prepared assemblies of sol and gel aggregates have been analyzed both by means of standard AFM Fourier software and a wavelet method. The deposited surfaces show a persistence to antipersistent crossover with a clay concentration dependent crossover length. It is concluded that the crossover length is associated with aggregate size, and further that the persistent roughness at small length scales signals near compact clusters of fractal dimension three, whereas the antipersistent roughness at large length scales signals a sedimentation process.

  18. Self-affine crossover length in a layered silicate deposit

    NASA Astrophysics Data System (ADS)

    Fossum, J. O.; Bergene, H. H.; Hansen, Alex; O'Rourke, B.; Manificat, G.

    2004-03-01

    Self-affine dehydrated colloidal deposits on fresh mica surfaces of the synthetic layered silicate 2:1 smectite clay laponite have been studied by means of atomic force microscopy (AFM). AFM images of these prepared assemblies of sol and gel aggregates have been analyzed both by means of standard AFM Fourier software and a wavelet method. The deposited surfaces show a persistence to antipersistent crossover with a clay concentration dependent crossover length. It is concluded that the crossover length is associated with aggregate size, and further that the persistent roughness at small length scales signals near compact clusters of fractal dimension three, whereas the antipersistent roughness at large length scales signals a sedimentation process.

  19. Affinity constants for small molecules from SPR competition experiments.

    PubMed

    de Mol, Nico J

    2010-01-01

    Direct assay of small molecules by SPR in general is troublesome and at least tedious procedures have to be applied. Competition experiments offer an attractive alternative. A small ligand known to bind to the analyte is immobilized on an SPR sensor surface, and the binding of the larger analyte in the presence of compounds under investigation in a concentration range is assayed. The resulting inhibition curves of the equilibrium SPR signal as function of the compound concentration can be analyzed to yield thermodynamic binding constants for the interaction in solution between analyte and the compounds under investigation. An additional advantage of this method is that series of compounds can be analyzed using the same sensor surface, so there is no immobilization needed for each compound. An adaptation of the method to analyze interactions with bivalent analytes (e.g., antibodies) is also included. Some observed different affinities in solution compared to that on the SPR surface are discussed.

  20. Vibrational photodetachment spectroscopy near the electron affinity of S2

    NASA Astrophysics Data System (ADS)

    Barrick, J. B.; Yukich, J. N.

    2016-02-01

    We have conducted laser photodetachment spectroscopy near the detachment threshold of the electron affinity of S2 in a 1.8-T field. The ions are prepared by dissociative electron attachment to carbonyl sulfide. The experiment is conducted in a Penning ion trap and with a narrow-band, tunable, Ti:sapphire laser. A hybrid model for photodetachment in an ion trap is fit to the data using the appropriate Franck-Condon factors. The observations reveal detachment from and to the first few vibrational levels of the anion and the neutral molecule, respectively. Evaporative cooling of the anion ensemble condenses the thermal distribution to the lowest initial vibrational states. The subsequent detachment spectroscopy yields results consistent with a vibrationally cooled anion population.

  1. Growth factors with heparin binding affinity in human synovial fluid

    SciTech Connect

    Hamerman, D.; Taylor, S.; Kirschenbaum, I.; Klagsbrun, M.; Raines, E.W.; Ross, R.; Thomas, K.A.

    1987-12-01

    Synovial effusions were obtained from the knees of 15 subjects with joint trauma, menisceal or ligamentous injury, or osteoarthritis. Heparin-Sepharose affinity chromatography of these synovial fluids revealed, in general, three major peaks of mitogenic activity as measured by incorporation of /sup 3/H-thymidine into 3T3 cells. Gradient elution patterns showed activities at 0.5M NaCl, which is characteristic of platelet derived growth factor, and at 1.1 M NaCl and 1.6M NaCl, indicative of acidic and basic fibroblast growth factors, respectively. The identities of these mitogenic fractions were confirmed by specific immunologic and receptor-binding assays. The presence of platelet derived, acidic and basic fibroblast growth factors in the synovial fluid may contribute to wound healing in the arthritic joint.

  2. The anatomy, affinity, and phylogenetic significance of Markuelia.

    PubMed

    Dong, Xi-Ping; Donoghue, Philip C J; Cunningham, John A; Liu, Jian-Bo; Cheng, Hong

    2005-01-01

    The fossil record provides a paucity of data on the development of extinct organisms, particularly for their embryology. The recovery of fossilized embryos heralds new insight into the evolution of development but advances are limited by an almost complete absence of phylogenetic constraint. Markuelia is an exception to this, known from cleavage and pre-hatchling stages as a vermiform and profusely annulated direct-developing bilaterian with terminal circumoral and posterior radial arrays of spines. Phylogenetic analyses have hitherto suggested assignment to stem-Scalidophora (phyla Kinorhyncha, Loricifera, Priapulida). We test this assumption with additional data and through the inclusion of additional taxa. The available evidence supports stem-Scalidophora affinity, leading to the conclusion that scalidophorans, cyclonerualians, and ecdysozoans are primitive direct developers, and the likelihood that scalidophorans are primitively metameric.

  3. DNA Shape Dominates Sequence Affinity in Nucleosome Formation

    NASA Astrophysics Data System (ADS)

    Freeman, Gordon S.; Lequieu, Joshua P.; Hinckley, Daniel M.; Whitmer, Jonathan K.; de Pablo, Juan J.

    2014-10-01

    Nucleosomes provide the basic unit of compaction in eukaryotic genomes, and the mechanisms that dictate their position at specific locations along a DNA sequence are of central importance to genetics. In this Letter, we employ molecular models of DNA and proteins to elucidate various aspects of nucleosome positioning. In particular, we show how DNA's histone affinity is encoded in its sequence-dependent shape, including subtle deviations from the ideal straight B-DNA form and local variations of minor groove width. By relying on high-precision simulations of the free energy of nucleosome complexes, we also demonstrate that, depending on DNA's intrinsic curvature, histone binding can be dominated by bending interactions or electrostatic interactions. More generally, the results presented here explain how sequence, manifested as the shape of the DNA molecule, dominates molecular recognition in the problem of nucleosome positioning.

  4. Advances in affinity ligand-functionalized nanomaterials for biomagnetic separation.

    PubMed

    Fields, Conor; Li, Peng; O'Mahony, James J; Lee, Gil U

    2016-01-01

    The downstream processing of proteins remains the most significant cost in protein production, and is largely attributed to rigorous chromatographic purification protocols, where the stringency of purity for biopharmaceutical products sometimes exceeds 99%. With an ever burgeoning biotechnology market, there is a constant demand for alternative purification methodologies, to ameliorate the dependence on chromatography, while still adhering to regulatory concerns over product purity and safety. In this article, we present an up-to-date view of bioseparation, with emphasis on magnetic separation and its potential application in the field. Additionally, we discuss the economic and performance benefits of synthetic ligands, in the form of peptides and miniaturized antibody fragments, compared to full-length antibodies. We propose that adoption of synthetic affinity ligands coupled with magnetic adsorbents, will play an important role in enabling sustainable bioprocessing in the future.

  5. Virus inactivation by protein denaturants used in affinity chromatography.

    PubMed

    Roberts, Peter L; Lloyd, David

    2007-10-01

    Virus inactivation by a number of protein denaturants commonly used in gel affinity chromatography for protein elution and gel recycling has been investigated. The enveloped viruses Sindbis, herpes simplex-1 and vaccinia, and the non-enveloped virus polio-1 were effectively inactivated by 0.5 M sodium hydroxide, 6 M guanidinium thiocyanate, 8 M urea and 70% ethanol. However, pH 2.6, 3 M sodium thiocyanate, 6 M guanidinium chloride and 20% ethanol, while effectively inactivating the enveloped viruses, did not inactivate polio-1. These studies demonstrate that protein denaturants are generally effective for virus inactivation but with the limitation that only some may inactivate non-enveloped viruses. The use of protein denaturants, together with virus reduction steps in the manufacturing process should ensure that viral cross contamination between manufacturing batches of therapeutic biological products is prevented and the safety of the product ensured.

  6. A High-Affinity Adenosine Kinase from Anopheles Gambiae

    SciTech Connect

    M Cassera; M Ho; E Merino; E Burgos; A Rinaldo-Matthis; S Almo; V Schramm

    2011-12-31

    Genome analysis revealed a mosquito orthologue of adenosine kinase in Anopheles gambiae (AgAK; the most important vector for the transmission of Plasmodium falciparum in Africa). P. falciparum are purine auxotrophs and do not express an adenosine kinase but rely on their hosts for purines. AgAK was kinetically characterized and found to have the highest affinity for adenosine (K{sub m} = 8.1 nM) of any known adenosine kinase. AgAK is specific for adenosine at the nucleoside site, but several nucleotide triphosphate phosphoryl donors are tolerated. The AgAK crystal structure with a bound bisubstrate analogue Ap{sub 4}A (2.0 {angstrom} resolution) reveals interactions for adenosine and ATP and the geometry for phosphoryl transfer. The polyphosphate charge is partly neutralized by a bound Mg{sup 2+} ion and an ion pair to a catalytic site Arg. The AgAK structure consists of a large catalytic core in a three-layer {alpha}/{beta}/{alpha} sandwich, and a small cap domain in contact with adenosine. The specificity and tight binding for adenosine arise from hydrogen bond interactions of Asn14, Leu16, Leu40, Leu133, Leu168, Phe168, and Thr171 and the backbone of Ile39 and Phe168 with the adenine ring as well as through hydrogen bond interactions between Asp18, Gly64, and Asn68 and the ribosyl 2'- and 3'-hydroxyl groups. The structure is more similar to that of human adenosine kinase (48% identical) than to that of AK from Toxoplasma gondii (31% identical). With this extraordinary affinity for AgAK, adenosine is efficiently captured and converted to AMP at near the diffusion limit, suggesting an important role for this enzyme in the maintenance of the adenine nucleotide pool. mRNA analysis verifies that AgAK transcripts are produced in the adult insects.

  7. Triazine dyes as inhibitors and affinity ligands of glycosyltransferases.

    PubMed

    Kamińska, J; Dziecioł, J; Kościelak, J

    1999-11-01

    The triazine dyes: Cibacron Blue 3GA, Reactive Red 120, Reactive Yellow 86, Reactive Green 19, Reactive Blue 4, Reactive Brown 10 inhibited the activity of a purified preparation of alpha1,6fucosyltransferase (GDP-L-fucose: N-acetyl beta-glucosaminide 6-alpha-L-fucosyltransferase, EC 2.4.1.68) from human blood platelets. Cibacron Blue 3GA and Reactive Red 120 were examined for the nature of the inhibition and both were found to be competitive inhibitors of the enzyme, with Ki = 11 microM and 2 microM, respectively. The two dyes inhibited also serum glycosyltransferases: alpha1,2fucosyltransferase (GDP-L-fucose: beta-D-galactosyl-R2-alpha-L-fucosyltransferase, EC 2.4.1.69), beta1,4galactosyltransferase (UDP-galactose: N-acetyl-D-glucosamine 4-beta-D-galactosyltransferase, EC 2.4.1.90) and beta1,3N-acetylglucosaminyltransferase (UDP-GlcNAc: 4-beta-D-galactosyl-D-glucose). Cibacron Blue 3GA was a more effective inhibitor of the glycosyltransferases that use UDP-linked sugar donors than Reactive Red 120 while the latter was a stronger inhibitor of the fucosyltransferases that use GDP-linked donor. All four glycosyltransferases could be affinity purified on Cibacron Blue 3GA-Agarose columns. The order of elution of glycosyltransferases from the columns with solutions of 0.25-1.0 M potassium iodide also depended upon the structure of nucleotide sugar donor, i.e. whether it contained UDP or GDP. Thus, triazine dyes should interact with the sugar donor binding sites of glycosyltransferases. The main advantages of the use of triazine dyes as affinity ligands for isolation of glycosyltransferases are their universal applicability regardless of enzyme specificity, low cost, and insensitivity to high concentration of other proteins present in the solution.

  8. Brain structure resolves the segmental affinity of anomalocaridid appendages.

    PubMed

    Cong, Peiyun; Ma, Xiaoya; Hou, Xianguang; Edgecombe, Gregory D; Strausfeld, Nicholas J

    2014-09-25

    Despite being among the most celebrated taxa from Cambrian biotas, anomalocaridids (order Radiodonta) have provoked intense debate about their affinities within the moulting-animal clade that includes Arthropoda. Current alternatives identify anomalocaridids as either stem-group euarthropods, crown-group euarthropods near the ancestry of chelicerates, or a segmented ecdysozoan lineage with convergent similarity to arthropods in appendage construction. Determining unambiguous affinities has been impeded by uncertainties about the segmental affiliation of anomalocaridid frontal appendages. These structures are variably homologized with jointed appendages of the second (deutocerebral) head segment, including antennae and 'great appendages' of Cambrian arthropods, or with the paired antenniform frontal appendages of living Onychophora and some Cambrian lobopodians. Here we describe Lyrarapax unguispinus, a new anomalocaridid from the early Cambrian Chengjiang biota, southwest China, nearly complete specimens of which preserve traces of muscles, digestive tract and brain. The traces of brain provide the first direct evidence for the segmental composition of the anomalocaridid head and its appendicular organization. Carbon-rich areas in the head resolve paired pre-protocerebral ganglia at the origin of paired frontal appendages. The ganglia connect to areas indicative of a bilateral pre-oral brain that receives projections from the eyestalk neuropils and compound retina. The dorsal, segmented brain of L. unguispinus reinforces an alliance between anomalocaridids and arthropods rather than cycloneuralians. Correspondences in brain organization between anomalocaridids and Onychophora resolve pre-protocerebral ganglia, associated with pre-ocular frontal appendages, as characters of the last common ancestor of euarthropods and onychophorans. A position of Radiodonta on the euarthropod stem-lineage implies the transformation of frontal appendages to another structure in crown

  9. Changing water affinity from hydrophobic to hydrophilic in hydrophobic channels.

    PubMed

    Ohba, Tomonori; Yamamoto, Shotaro; Kodaira, Tetsuya; Hata, Kenji

    2015-01-27

    The behavior of water at hydrophobic interfaces can play a significant role in determining chemical reaction outcomes and physical properties. Carbon nanotubes and aluminophosphate materials have one-dimensional hydrophobic channels, which are entirely surrounded by hydrophobic interfaces. Unique water behavior was observed in such hydrophobic channels. In this article, changes in the water affinity in one-dimensional hydrophobic channels were assessed using water vapor adsorption isotherms at 303 K and grand canonical Monte Carlo simulations. Hydrophobic behavior of water adsorbed in channels wider than 3 nm was observed for both adsorption and desorption processes, owing to the hydrophobic environment. However, water showed hydrophilic properties in both adsorption and desorption processes in channels narrower than 1 nm. In intermediate-sized channels, the hydrophobic properties of water during the adsorption process were seen to transition to hydrophilic behavior during the desorption process. Hydrophilic properties in the narrow channels for both adsorption and desorption processes are a result of the relatively strong water-channel interactions (10-15 kJ mol(-1)). In the 2-3 nm channels, the water-channel interaction energy of 4-5 kJ mol(-1) was comparable to the thermal translational energy. The cohesive water interaction was approximately 35 kJ mol(-1), which was larger than the others. Thus, the water affinity change in the 2-3 nm channels for the adsorption and desorption processes was attributed to weak water-channel interactions and strong cohesive interactions. These results are inherently important to control the properties of water in hydrophobic environments.

  10. Electron affinity of cubic boron nitride terminated with vanadium oxide

    SciTech Connect

    Yang, Yu; Sun, Tianyin; Shammas, Joseph; Hao, Mei; Nemanich, Robert J.; Kaur, Manpuneet

    2015-10-28

    A thermally stable negative electron affinity (NEA) for a cubic boron nitride (c-BN) surface with vanadium-oxide-termination is achieved, and its electronic structure was analyzed with in-situ photoelectron spectroscopy. The c-BN films were prepared by electron cyclotron resonance plasma-enhanced chemical vapor deposition employing BF{sub 3} and N{sub 2} as precursors. Vanadium layers of ∼0.1 and 0.5 nm thickness were deposited on the c-BN surface in an electron beam deposition system. Oxidation of the metal layer was achieved by an oxygen plasma treatment. After 650 °C thermal annealing, the vanadium oxide on the c-BN surface was determined to be VO{sub 2}, and the surfaces were found to be thermally stable, exhibiting an NEA. In comparison, the oxygen-terminated c-BN surface, where B{sub 2}O{sub 3} was detected, showed a positive electron affinity of ∼1.2 eV. The B{sub 2}O{sub 3} evidently acts as a negatively charged layer introducing a surface dipole directed into the c-BN. Through the interaction of VO{sub 2} with the B{sub 2}O{sub 3} layer, a B-O-V layer structure would contribute a dipole between the O and V layers with the positive side facing vacuum. The lower enthalpy of formation for B{sub 2}O{sub 3} is favorable for the formation of the B-O-V layer structure, which provides a thermally stable surface dipole and an NEA surface.

  11. Rejuvenation of metallic glasses by non-affine thermal strain.

    PubMed

    Ketov, S V; Sun, Y H; Nachum, S; Lu, Z; Checchi, A; Beraldin, A R; Bai, H Y; Wang, W H; Louzguine-Luzgin, D V; Carpenter, M A; Greer, A L

    2015-08-13

    When a spatially uniform temperature change is imposed on a solid with more than one phase, or on a polycrystal of a single, non-cubic phase (showing anisotropic expansion-contraction), the resulting thermal strain is inhomogeneous (non-affine). Thermal cycling induces internal stresses, leading to structural and property changes that are usually deleterious. Glasses are the solids that form on cooling a liquid if crystallization is avoided--they might be considered the ultimate, uniform solids, without the microstructural features and defects associated with polycrystals. Here we explore the effects of cryogenic thermal cycling on glasses, specifically metallic glasses. We show that, contrary to the null effect expected from uniformity, thermal cycling induces rejuvenation, reaching less relaxed states of higher energy. We interpret these findings in the context that the dynamics in liquids become heterogeneous on cooling towards the glass transition, and that there may be consequent heterogeneities in the resulting glasses. For example, the vibrational dynamics of glassy silica at long wavelengths are those of an elastic continuum, but at wavelengths less than approximately three nanometres the vibrational dynamics are similar to those of a polycrystal with anisotropic grains. Thermal cycling of metallic glasses is easily applied, and gives improvements in compressive plasticity. The fact that such effects can be achieved is attributed to intrinsic non-uniformity of the glass structure, giving a non-uniform coefficient of thermal expansion. While metallic glasses may be particularly suitable for thermal cycling, the non-affine nature of strains in glasses in general deserves further study, whether they are induced by applied stresses or by temperature change.

  12. The high-affinity maltose switch MBP317-347 has low affinity for glucose: implications for targeting tumors with metabolically directed enzyme prodrug therapy.

    PubMed

    Valdes, Gilmer; Schulte, Reinhard W; Ostermeier, Marc; Iwamoto, Keisuke S

    2014-03-01

    Development of agents with high affinity and specificity for tumor-specific markers is an important goal of molecular-targeted therapy. Here, we propose a shift in paradigm using a strategy that relies on low affinity for fundamental metabolites found in different concentrations in cancerous and non-cancerous tissues: glucose and lactate. A molecular switch, MBP317-347, originally designed to be a high-affinity switch for maltose and maltose-like polysaccharides, was demonstrated to be a low-affinity switch for glucose, that is, able to be activated by high concentrations (tens of millimolar) of glucose. We propose that such a low-affinity glucose switch could be used as a proof of concept for a new prodrug therapy strategy denominated metabolically directed enzyme prodrug therapy (MDEPT) where glucose or, preferably, lactate serves as the activator. Accordingly, considering the typical differential concentrations of lactate found in tumors and in healthy tissues, a low-affinity lactate-binding switch analogous to the low-affinity glucose-binding switch MBP317-347 would be an order of magnitude more active in tumors than in normal tissues and therefore can work as a differential activator of anticancer drugs in tumors.

  13. Cloning and functional characterization of the high-affinity K+ transporter HAK1 of pepper.

    PubMed

    Martínez-Cordero, M Angeles; Martínez, Vicente; Rubio, Francisco

    2004-10-01

    High-affinity K+ uptake in plants plays a crucial role in K+ nutrition and different systems have been postulated to contribute to the high-affinity K+ uptake. The results presented here with pepper (Capsicum annum) demonstrate that a HAK1-type transporter greatly contributes to the high-affinity K+ uptake observed in roots. Pepper plants starved of K+ for 3 d showed high-affinity K+ uptake (Km of 6 microM K+) that was very sensitive to NH and their roots expressed a high-affinity K+ transporter, CaHAK1, which clusters in group I of the KT/HAK/KUP family of transporters. When expressed in yeast ( Saccharomyces cerevisiae ), CaHAK1 mediated high-affinity K+ and Rb+ uptake with Km values of 3.3 and 1.9 microM, respectively. Rb+ uptake was competitively inhibited by micromolar concentrations of NH and Cs+, and by millimolar concentrations of Na+.

  14. Describing high-dimensional dynamics with low-dimensional piecewise affine models: applications to renewable energy.

    PubMed

    Hirata, Yoshito; Aihara, Kazuyuki

    2012-06-01

    We introduce a low-dimensional description for a high-dimensional system, which is a piecewise affine model whose state space is divided by permutations. We show that the proposed model tends to predict wind speeds and photovoltaic outputs for the time scales from seconds to 100 s better than by global affine models. In addition, computations using the piecewise affine model are much faster than those of usual nonlinear models such as radial basis function models.

  15. Data detailing the platelet acetyl-lysine proteome.

    PubMed

    Aslan, Joseph E; David, Larry L; McCarty, Owen J T

    2015-12-01

    Here we detail proteomics data that describe the acetyl-lysine proteome of blood platelets (Aslan et al., 2015 [1]). An affinity purification - mass spectrometry (AP-MS) approach was used to identify proteins modified by Nε-lysine acetylation in quiescent, washed human platelets. The data provide insights into potential regulatory mechanisms of platelet function mediated by protein lysine acetylation. Additionally, as platelets are anucleate and lack histone proteins, they offer a unique and valuable system to study the regulation of cytosolic proteins by lysine acetylation. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (Vizcaino et al., 2014 [2]) via with PRIDE partner repository with the dataset identifier PXD002332.

  16. Interlaboratory reproducibility of large-scale human protein-complex analysis by standardized AP-MS.

    PubMed

    Varjosalo, Markku; Sacco, Roberto; Stukalov, Alexey; van Drogen, Audrey; Planyavsky, Melanie; Hauri, Simon; Aebersold, Ruedi; Bennett, Keiryn L; Colinge, Jacques; Gstaiger, Matthias; Superti-Furga, Giulio

    2013-04-01

    The characterization of all protein complexes of human cells under defined physiological conditions using affinity purification-mass spectrometry (AP-MS) is a highly desirable step in the quest to understand the phenotypic effects of genomic information. However, such a challenging goal has not yet been achieved, as it requires reproducibility of the experimental workflow and high data consistency across different studies and laboratories. We systematically investigated the reproducibility of a standardized AP-MS workflow by performing a rigorous interlaboratory comparative analysis of the interactomes of 32 human kinases. We show that it is possible to achieve high interlaboratory reproducibility of this standardized workflow despite differences in mass spectrometry configurations and subtle sample preparation-related variations and that combination of independent data sets improves the approach sensitivity, resulting in even more-detailed networks. Our analysis demonstrates the feasibility of obtaining a high-quality map of the human protein interactome with a multilaboratory project.

  17. Intermediate affinity and potency of clozapine and low affinity of other neuroleptics and of antidepressants at H3 receptors.

    PubMed

    Kathmann, M; Schlicker, E; Göthert, M

    1994-12-01

    It was the aim of the present study to determine the affinities of four neuroleptics and five antidepressants for histamine H3 receptors. In rat brain cortex membranes, the specifically bound [3H]-N alpha-methylhistamine was monophasically displaced by clozapine (pKi 6.15). The other drugs did not completely displace the radioligand even at 100 microM; the pKi values were: haloperidol (4.91); sulpiride (4.73); amitriptyline (4.56); desipramine (4.15); levomepromazine (4.14); fluovoxamine (4.13); maprotiline (4.09); moclobemide (< 4.0). The effect of clozapine was further examined in a functional H3 receptor model, i.e., in superfused mouse brain cortex slices preincubated with [3H]-noradrenaline. The electrically evoked tritium overflow was not affected by clozapine 0.5-32 microM. However, clozapine shifted the concentration-response curve of histamine for its inhibitory effect on the evoked overflow to the right, but did not affect the maximum effect of histamine. The Schild plot yielded a pA2 value of 6.33. In conclusion, clozapine shows an intermediate affinity and potency (as a competitive antagonist) at H3 receptors. The Ki value of clozapine at H3 receptors resembles its Ki value at D2 receptors (the target of the classical neuroleptics), but is higher than its Ki values at D4, 5-HT2 or muscarinic acetylcholine receptors, which according to current hypotheses, might be involved in the atypical profile of clozapine.

  18. A study of the non-covalent interaction between flavonoids and DNA triplexes by electrospray ionization mass spectrometry

    NASA Astrophysics Data System (ADS)

    Wan, Cuihong; Cui, Meng; Song, Fengrui; Liu, Zhiqiang; Liu, Shuying

    2009-06-01

    The binding interactions of 22 flavonoids (9 aglycones and 13 glycosides) with DNA triplexes were investigated using electrospray ionization mass spectrometry (ESI-MS). The results revealed that the hydroxyl positions of aglycones, the locations and numbers of saccharide, as well as the aglycone skeletons play roles in the triplex-binding properties of flavonoids. The presence of 3-OH, or 3'-OH, or replacement of 4'-OH with methoxy group in aglycones decreased the fraction of bound DNA sharply. Flavonoid glycosides exhibit higher binding affinities towards the DNA triplexes than their aglycone counterparts. Glycosylations of flavones at the 8-C position and isoflavones at the 7-O position show higher binding affinities than those on the other positions of ring A of aglycones. Glycosylation with a disaccharide on C3 position of flavonol results in higher binding affinity than that with monosaccharide. Flexibility of the ring B is favorable for its interaction with DNA triplex. According to sustained off-resonance irradiation collision-induced dissociation (SORI-CID) experiments, glycosylation and non-planarity of flavonoid aglycones lead to different dissociation pathways of the flavonoid/triplex complexes. The differences between dissociation patterns suggest different DNA-binding modes or DNA-binding affinities. Although the exact binding geometry of the flavonoid-triplex complexes cannot be specified, the results may be helpful for understanding the triplex-binding properties of flavonoids and give a clue to design of triplex-binding ligands.

  19. Advances in protein complex analysis using mass spectrometry

    PubMed Central

    Gingras, Anne-Claude; Aebersold, Ruedi; Raught, Brian

    2005-01-01

    Proteins often function as components of larger complexes to perform a specific function, and formation of these complexes may be regulated. For example, intracellular signalling events often require transient and/or regulated protein–protein interactions for propagation, and protein binding to a specific DNA sequence, RNA molecule or metabolite is often regulated to modulate a particular cellular function. Thus, characterizing protein complexes can offer important insights into protein function. This review describes recent important advances in mass spectrometry (MS)-based techniques for the analysis of protein complexes. Following brief descriptions of how proteins are identified using MS, and general protein complex purification approaches, we address two of the most important issues in these types of studies: specificity and background protein contaminants. Two basic strategies for increasing specificity and decreasing background are presented: whereas (1) tandem affinity purification (TAP) of tagged proteins of interest can dramatically improve the signal-to-noise ratio via the generation of cleaner samples, (2) stable isotopic labelling of proteins may be used to discriminate between contaminants and bona fide binding partners using quantitative MS techniques. Examples, as well as advantages and disadvantages of each approach, are presented. PMID:15611014

  20. Synthetic studies of neoclerodane diterpenoids from Salvia splendens and evaluation of Opioid Receptor affinity.

    PubMed

    Fontana, Gianfranco; Savona, Giuseppe; Rodríguez, Benjamín; Dersch, Christina M; Rothman, Richard B; Prisinzano, Thomas E

    2008-12-20

    Salvinorin A (1), a neoclerodane diterpene from the hallucinogenic mint Salvia divinorum, is the only known non-nitrogenous and specific kappa-opioid agonist. Several structural congeners of 1 isolated from Salvia splendens (2 - 8) together with a series of semisynthetic derivatives (9 - 24), some of which possess a pyrazoline structural moiety (9, 19 - 22), have been tested for affinity at human mu, delta, and kappa opioid receptors. None of these compounds showed high affinity binding to these receptors. However, 10 showed modest affinity for kappa receptors suggesting other naturally neoclerodanes from different Salvia species may possess opioid affinity.

  1. Affinity enhancement of an in vivo matured therapeutic antibody using structure-based computational design.

    PubMed

    Clark, Louis A; Boriack-Sjodin, P Ann; Eldredge, John; Fitch, Christopher; Friedman, Bethany; Hanf, Karl J M; Jarpe, Matthew; Liparoto, Stefano F; Li, You; Lugovskoy, Alexey; Miller, Stephan; Rushe, Mia; Sherman, Woody; Simon, Kenneth; Van Vlijmen, Herman

    2006-05-01

    Improving the affinity of a high-affinity protein-protein interaction is a challenging problem that has practical applications in the development of therapeutic biomolecules. We used a combination of structure-based computational methods to optimize the binding affinity of an antibody fragment to the I-domain of the integrin VLA1. Despite the already high affinity of the antibody (Kd approximately 7 nM) and the moderate resolution (2.8 A) of the starting crystal structure, the affinity was increased by an order of magnitude primarily through a decrease in the dissociation rate. We determined the crystal structure of a high-affinity quadruple mutant complex at 2.2 A. The structure shows that the design makes the predicted contacts. Structural evidence and mutagenesis experiments that probe a hydrogen bond network illustrate the importance of satisfying hydrogen bonding requirements while seeking higher-affinity mutations. The large and diverse set of interface mutations allowed refinement of the mutant binding affinity prediction protocol and improvement of the single-mutant success rate. Our results indicate that structure-based computational design can be successfully applied to further improve the binding of high-affinity antibodies.

  2. Affinity functions: recognizing essential parameters in fuzzy connectedness based image segmentation

    NASA Astrophysics Data System (ADS)

    Ciesielski, Krzysztof C.; Udupa, Jayaram K.

    2009-02-01

    Fuzzy connectedness (FC) constitutes an important class of image segmentation schemas. Although affinity functions represent the core aspect (main variability parameter) of FC algorithms, they have not been studied systematically in the literature. In this paper, we present a thorough study to fill this gap. Our analysis is based on the notion of equivalent affinities: if any two equivalent affinities are used in the same FC schema to produce two versions of the algorithm, then these algorithms are equivalent in the sense that they lead to identical segmentations. We give a complete characterization of the affinity equivalence and show that many natural definitions of affinity functions and their parameters used in the literature are redundant in the sense that different definitions and values of such parameters lead to equivalent affinities. We also show that two main affinity types - homogeneity based and object feature based - are equivalent, respectively, to the difference quotient of the intensity function and Rosenfeld's degree of connectivity. In addition, we demonstrate that any segmentation obtained via relative fuzzy connectedness (RFC) algorithm can be viewed as segmentation obtained via absolute fuzzy connectedness (AFC) algorithm with an automatic and adaptive threshold detection. We finish with an analysis of possible ways of combining different component affinities that result in non equivalent affinities.

  3. Neuroscience and Accelerator Mass Spectrometry

    SciTech Connect

    Palmblad, M N; Buchholz, B A; Hillegonds, D J; Vogel, J S

    2004-08-02

    Accelerator mass spectrometry (AMS) is a mass spectrometric method for quantifying rare isotopes. It has had great impact in geochronology and archaeology and is now being applied in biomedicine. AMS measures radioisotopes such as {sup 3}H, {sup 14}C, {sup 26}Al, {sup 36}Cl and {sup 41}Ca, with zepto- or attomole sensitivity and high precision and throughput, enabling safe human pharmacokinetic studies involving: microgram doses, agents having low bioavailability, or toxicology studies where administered doses must be kept low (<1 {micro}g/kg). It is used to study long-term pharmacokinetics, to identify biomolecular interactions, to determine chronic and low-dose effects or molecular targets of neurotoxic substances, to quantify transport across the blood-brain barrier and to resolve molecular turnover rates in the human brain on the timescale of decades. We will here review how AMS is applied in neurotoxicology and neuroscience.

  4. Electrophoresis-mass spectrometry probe

    DOEpatents

    Andresen, Brian D.; Fought, Eric R.

    1987-01-01

    The invention involves a new technique for the separation of complex mixtures of chemicals, which utilizes a unique interface probe for conventional mass spectrometers which allows the electrophoretically separated compounds to be analyzed in real-time by a mass spectrometer. This new chemical analysis interface, which couples electrophoresis with mass spectrometry, allows complex mixtures to be analyzed very rapidly, with much greater specificity, and with greater sensitivity. The interface or probe provides a means whereby large and/or polar molecules in complex mixtures to be completely characterized. The preferred embodiment of the probe utilizes a double capillary tip which allows the probe tip to be continually wetted by the buffer, which provides for increased heat dissipation, and results in a continually operating interface which is more durable and electronically stable than the illustrated single capillary tip probe interface.

  5. Electrophoresis-mass spectrometry probe

    DOEpatents

    Andresen, B.D.; Fought, E.R.

    1987-11-10

    The invention involves a new technique for the separation of complex mixtures of chemicals, which utilizes a unique interface probe for conventional mass spectrometers which allows the electrophoretically separated compounds to be analyzed in real-time by a mass spectrometer. This new chemical analysis interface, which couples electrophoresis with mass spectrometry, allows complex mixtures to be analyzed very rapidly, with much greater specificity, and with greater sensitivity. The interface or probe provides a means whereby large and/or polar molecules in complex mixtures to be completely characterized. The preferred embodiment of the probe utilizes a double capillary tip which allows the probe tip to be continually wetted by the buffer, which provides for increased heat dissipation, and results in a continually operating interface which is more durable and electronically stable than the illustrated single capillary tip probe interface. 8 figs.

  6. Neuroscience and accelerator mass spectrometry.

    PubMed

    Palmblad, Magnus; Buchholz, Bruce A; Hillegonds, Darren J; Vogel, John S

    2005-02-01

    Accelerator mass spectrometry (AMS) is a mass spectrometric method for quantifying rare isotopes. It has had a great impact in geochronology and archaeology and is now being applied in biomedicine. AMS measures radioisotopes such as 3H, 14C, 26Al, 36Cl and 41Ca, with zepto- or attomole sensitivity and high precision and throughput, allowing safe human pharmacokinetic studies involving microgram doses, agents having low bioavailability or toxicology studies where administered doses must be kept low (<1 microg kg(-1)). It is used to study long-term pharmacokinetics, to identify biomolecular interactions, to determine chronic and low-dose effects or molecular targets of neurotoxic substances, to quantify transport across the blood-brain barrier and to resolve molecular turnover rates in the human brain on the time-scale of decades. We review here how AMS is applied in neurotoxicology and neuroscience.

  7. Laser Ablation Molecular Isotopic Spectrometry

    NASA Astrophysics Data System (ADS)

    Russo, Richard E.; Bol'shakov, Alexander A.; Mao, Xianglei; McKay, Christopher P.; Perry, Dale L.; Sorkhabi, Osman

    2011-02-01

    A new method of performing optical isotopic analysis of condensed samples in ambient air and at ambient pressure has been developed: Laser Ablation Molecular Isotopic Spectrometry (LAMIS). The technique uses radiative transitions from molecular species either directly vaporized from a sample or formed by associative mechanisms of atoms or ions in a laser ablation plume. This method is an advanced modification of a known atomic emission technique called laser-induced breakdown spectroscopy (LIBS). The new method — LAMIS — can determine not only chemical composition but also isotopic ratios of elements in the sample. Isotopic measurements are enabled by significantly larger isotopic shifts found in molecular spectra relative to atomic spectra. Analysis can be performed from a distance and in real time. No sample preparation or pre-treatment is required. Detection of the isotopes of hydrogen, boron, carbon, and oxygen are discussed to illustrate the technique.

  8. High Affinity Binding of Indium and Ruthenium Ions by Gastrins.

    PubMed

    Baldwin, Graham S; George, Graham N; Pushie, M Jake

    2015-01-01

    The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS) spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga3+ were fitted by a 2 site model with dissociation constants (Kd) of 3.3 x 10-7 and 1.1 x 10-6 M. Although the absorption of gastrin did not change upon the addition of In3+ ions, the changes in absorbance on Fe3+ ion binding in the presence of indium ions were fitted by a 2 site model with Kd values for In3+ of 6.5 x 10-15 and 1.7 x 10-7 M. Similar results were obtained with Ru3+ ions, although the Kd values for Ru3+ of 2.6 x 10-13 and 1.2 x 10-5 M were slightly larger than observed for In3+. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0-3.3 Å, the Ru complex clearly demonstrated a short range Ru-Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In3+ or Ru3+ ions, and that the affinity of the first site for In3+ or Ru3+ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy.

  9. The phylogeny of varanoid lizards and the affinities of snakes

    PubMed Central

    Lee, M. S. Y.

    1997-01-01

    Evidence that platynotan squamates (living varanoid lizards, snakes and their fossil relatives) are monophyletic is presented. Evolutionary relationships within this group are then ascertained through a cladistic analysis of 144 osteological characters. Mosasauroids (aigialosaurs and mosasaurs), a group of large marine lizards, are identified as the nearest relatives of snakes, thus resolving the long-standing problem of snake affinities. The mosasauroid–snake clade (Pythonomorpha) is corroborated by 40 derived characters, including recumbent replacement teeth, thecodonty, four or fewer premaxillary teeth, supratemporal–prootic contact, free mandibular tips, crista circumfenestralis, straight vertical splenio-angular joint, loss of posterior ramus of the coronoid, reduced basipterygoid processes, reduced interpterygoid vacuity, zygosphene–zygantral articulations, and absence of epiphyses on the axial skeleton and skull. After mosasauroids, the next closest relatives of snakes are varanids (Varanus, Saniwa and Saniwides) and lanthanotids (Lanthanotus and Cherminotus). Derived features uniting varanids and lanthanotids include nine cervical vertebrae and three or fewer pairs of sternal ribs. The varanid–lanthanotid–pythonomorph clade, here termed Thecoglossa, is supported by features such as the anteriorly positioned basal tubera, and the loss of the second epibranchial. Successive outgroups to thecoglossans are Telmasaurus, an unresolved polytomy (Estesia, Gobidermatidae and Helodermatidae), Paravaranus and Proplatynota. The 'necrosaurs' are demonstrated to be an artificial (polyphyletic) assemblage of primitive platynotans that are not particularly closely related to each other. Snakes are presumed to have evolved from small, limbless, burrowing lizards and the inability of previous analyses to resolve the affinities of snakes has been attributed to extensive convergence among the numerous lineages of such lizards. The present study contradicts this claim

  10. Higher Nucleoporin-Importinβ Affinity at the Nuclear Basket Increases Nucleocytoplasmic Import

    PubMed Central

    Azimi, Mohammad; Mofrad, Mohammad R. K.

    2013-01-01

    Several in vitro studies have shown the presence of an affinity gradient in nuclear pore complex proteins for the import receptor Importinβ, at least partially contributing to nucleocytoplasmic transport, while others have historically argued against the presence of such a gradient. Nonetheless, the existence of an affinity gradient has remained an uncharacterized contributing factor. To shed light on the affinity gradient theory and better characterize how the existence of such an affinity gradient between the nuclear pore and the import receptor may influence the nucleocytoplasmic traffic, we have developed a general-purpose agent based modeling (ABM) framework that features a new method for relating rate constants to molecular binding and unbinding probabilities, and used our ABM approach to quantify the effects of a wide range of forward and reverse nucleoporin-Importinβ affinity gradients. Our results indicate that transport through the nuclear pore complex is maximized with an effective macroscopic affinity gradient of 2000 µM, 200 µM and 10 µM in the cytoplasmic, central channel and nuclear basket respectively. The transport rate at this gradient is approximately 10% higher than the transport rate for a comparable pore lacking any affinity gradient, which has a peak transport rate when all nucleoporins have an affinity of 200 µM for Importinβ. Furthermore, this optimal ratio of affinity gradients is representative of the ratio of affinities reported for the yeast nuclear pore complex – suggesting that the affinity gradient seen in vitro is highly optimized. PMID:24282617

  11. Short-term desensitization of muscarinic cholinergic receptors in mouse neuroblastoma cells: selective loss of agonist low-affinity and pirenzepine high-affinity binding sites

    SciTech Connect

    Cioffi, C.L.; el-Fakahany, E.E.

    1986-09-01

    The effects of brief incubation with carbamylcholine on subsequent binding of (/sup 3/H)N-methylscopolamine were investigated in mouse neuroblastoma cells (clone N1E-115). This treatment demonstrated that the muscarinic receptors in this neuronal clone can be divided into two types; one which is readily susceptible to regulation by receptor agonists, whereas the other is resistant in this regard. In control cells, both pirenzepine and carbamylcholine interacted with high- and low-affinity subsets of muscarinic receptors. Computer-assisted analysis of the competition between pirenzepine and carbamylcholine with (/sup 3/H)N-methylscopolamine showed that the receptor sites remaining upon desensitization are composed mainly of pirenzepine low-affinity and agonist high-affinity binding sites. Furthermore, there was an excellent correlation between the ability of various muscarinic receptor agonists to induce a decrease in consequent (/sup 3/H)N-methylscopolamine binding and their efficacy in stimulating cyclic GMP synthesis in these cells. Thus, only the agonists that are known to recognize the receptor's low-affinity conformation in order to elicit increases in cyclic GMP levels were capable of diminishing ligand binding. Taken together, our present results suggest that the receptor population that is sensitive to regulation by agonists includes both the pirenzepine high-affinity and the agonist low-affinity receptor binding states. In addition, the sensitivity of these receptor subsets to rapid regulation by agonists further implicates their involvement in desensitization of muscarinic receptor-mediated cyclic GMP formation.

  12. A new alpha-globin variant with increased oxygen affinity in a Swiss family: Hb Frauenfeld [alpha 138(H21)Ser-->Phe, TCC>TTC (alpha 2)].

    PubMed

    Hochuli, Michel; Zurbriggen, Karin; Schmid, Marlis; Speer, Oliver; Rochat, Philippe; Frauchiger, Beat; Kleinert, Peter; Schmugge, Markus; Troxler, Heinz

    2009-01-01

    A new alpha-globin mutation [alpha 138(H21)Ser-->Phe] was found in a 55-year-old male proband with an erythrocytosis known since his youth. Cation exchange high performance liquid chromatography (HPLC) revealed an additional peak eluting slightly before Hb A indicating the presence of a variant. The peak area of the variant was approximately one-third that of Hb A suggesting an alpha-globin variant. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis confirmed the mutation at the protein level. The variant is also detectable with isoelectric focusing and reversed phase HPLC. DNA analysis revealed a heterozygous sequence mutation at codon 138 of the alpha2 gene. A C>T transition at the second nucleotide of the codon indicated a Ser-->Phe exchange. The variant showed increased oxygen affinity and was named Hb Frauenfeld.

  13. High-resolution mapping of the protein interaction network for the human transcription machinery and affinity purification of RNA polymerase II-associated complexes

    PubMed Central

    Cloutier, Philippe; Al-Khoury, Racha; Lavallée-Adam, Mathieu; Faubert, Denis; Jiang, Heng; Poitras, Christian; Bouchard, Annie; Forget, Diane; Blanchette, Mathieu; Coulombe, Benoit

    2015-01-01

    Thirty years of research on gene transcription has uncovered a myriad of factors that regulate, directly or indirectly, the activity of RNA polymerase II (RNAPII) during mRNA synthesis. Yet many regulatory factors remain to be discovered. Using protein affinity purification coupled to mass spectrometry (AP-MS), we recently unraveled a high-density interaction network formed by RNAPII and its accessory factors from the soluble fraction of human cell extracts. Validation of the dataset using a machine learning approach trained to minimize the rate of false positives and false negatives yielded a high-confidence dataset and uncovered novel interactors that regulate the RNAPII transcription machinery, including a new protein assembly we named the RNAPII-Associated Protein 3 (RPAP3) complex. PMID:19450687

  14. Characterization of microbial siderophores by mass spectrometry.

    PubMed

    Pluháček, Tomáš; Lemr, Karel; Ghosh, Dipankar; Milde, David; Novák, Jiří; Havlíček, Vladimír

    2016-01-01

    Siderophores play important roles in microbial iron piracy, and are applied as infectious disease biomarkers and novel pharmaceutical drugs. Inductively coupled plasma and molecular mass spectrometry (ICP-MS) combined with high resolution separations allow characterization of siderophores in complex samples taking advantages of mass defect data filtering, tandem mass spectrometry, and iron-containing compound quantitation. The enrichment approaches used in siderophore analysis and current ICP-MS technologies are reviewed. The recent tools for fast dereplication of secondary metabolites and their databases are reported. This review on siderophores is concluded with their recent medical, biochemical, geochemical, and agricultural applications in mass spectrometry context.

  15. Fast left ventricle tracking using localized anatomical affine optical flow.

    PubMed

    Queirós, Sandro; Vilaça, João L; Morais, Pedro; Fonseca, Jaime C; D'hooge, Jan; Barbosa, Daniel

    2017-02-16

    In daily clinical cardiology practice, left ventricle (LV) global and regional function assessment is crucial for disease diagnosis, therapy selection and patient follow-up. Currently, this is still a time-consuming task, spending valuable human resources. In this work, a novel fast methodology for automatic LV tracking is proposed based on localized anatomically constrained affine optical flow. This novel method can be combined to previously proposed segmentation frameworks or manually delineated surfaces at an initial frame to obtain fully delineated datasets and, thus, assess both global and regional myocardial function. Its feasibility and accuracy was investigated in three distinct public databases, namely in realistically simulated 3D ultrasound (US), clinical 3D echocardiography and clinical cine cardiac magnetic resonance (CMR) images. The method showed accurate tracking results in all databases, proving its applicability and accuracy for myocardial function assessment. Moreover, when combined to previous state-of-the-art segmentation frameworks, it outperformed previous tracking strategies in both 3D US and CMR data, automatically computing relevant cardiac indices with smaller biases and narrower limits of agreement compared to reference indices. Simultaneously, the proposed localized tracking method showed to be suitable for online processing, even for 3D motion assessment. Importantly, although here evaluated for LV tracking only, this novel methodology is applicable for tracking of other target structures with minimal adaptations. This article is protected by copyright. All rights reserved.

  16. Experimental Immunization Based on Plasmodium Antigens Isolated by Antibody Affinity

    PubMed Central

    Kamali, Ali N.; Marín-García, Patricia; Azcárate, Isabel G.; Puyet, Antonio; Diez, Amalia; Bautista, José M.

    2015-01-01

    Vaccines blocking malaria parasites in the blood-stage diminish mortality and morbidity caused by the disease. Here, we isolated antigens from total parasite proteins by antibody affinity chromatography to test an immunization against lethal malaria infection in a murine model. We used the sera of malaria self-resistant ICR mice to lethal Plasmodium yoelii yoelii 17XL for purification of their IgGs which were subsequently employed to isolate blood-stage parasite antigens that were inoculated to immunize BALB/c mice. The presence of specific antibodies in vaccinated mice serum was studied by immunoblot analysis at different days after vaccination and showed an intensive immune response to a wide range of antigens with molecular weight ranging between 22 and 250 kDa. The humoral response allowed delay of the infection after the inoculation to high lethal doses of P. yoelii yoelii 17XL resulting in a partial protection against malaria disease, although final survival was managed in a low proportion of challenged mice. This approach shows the potential to prevent malaria disease with a set of antigens isolated from blood-stage parasites. PMID:26539558

  17. Surface-modified magnetic colloids for affinity adsorption of immunoglobulins

    NASA Astrophysics Data System (ADS)

    Martins, Fernanda; Pinho, Samantha C.; Zollner, Terezinha C. A.; Zollner, Ricardo L.; de Cuyper, Marcel; Santana, Maria Helena A.

    This work describes the preparation, characterization and in vitro adsorption tests of surface-modified magnetoliposomes for affinity binding of (i) anticardiolipin (isotype G) antibodies and (ii) specific isotype E antibodies generated by hypersensitivity reactions in humans with respiratory allergy. In the first case, cardiolipin embedded in the bilayer of magnetoliposomes was used as specific ligand. In the second case, antigenic proteins present in an extract of Dermatophagoids pteronyssinus and Blomia tropicalis mites were covalently coupled on the surface of magnetoliposomes via a diglycolic spacer arm, and used as specific ligands for IgE. Antibody adsorption was performed in a high-gradient magnetophoresis system, using either sera of healthy individuals or a pool of sera from autoimmune or allergic patients. The selectivity and capacity of the system were quantified by a frontal analysis in a capillary column, and by constructing breakthrough curves. The results show that the highest yield and selectivity were obtained if the ligand was extended into the aqueous layer surrounding the magnetoliposome surface. A 100% selectivity was obtained for adsorption of specific IgE, and 8% for IgG. These results demonstrate the potentialities of both types of surface-modified magnetic biocolloids in the field of in vitro diagnosis tests for allergic or autoimmune conditions.

  18. Preparation of phenylboronate affinity rigid monolith with macromolecular porogen.

    PubMed

    Li, Xiang-Jie; Jia, Man; Zhao, Yong-Xin; Liu, Zhao-Sheng; Akber Aisa, Haji

    2016-03-18

    Boronate-affinity monolithic column was first prepared via polystyrene (PS) as porogen in this work. The monolithic polymer was synthetized using 4-vinylphenylboronic acid (4-VPBA) as functional monomer, ethylene glycol dimethacrylate (EDMA) as crosslinker monomer, and a mixture of PS solution in tetrahydrofuran, the linear macromolecular porogen, and toluene as porogen. Isoquercitrin (ISO) and hyperoside (HYP), isomer diol flavonoid glycosides, can be baseline separated on the poly(VPBA-co-EDMA) monolith. The effect of polymerization variables on the selectivity factor, e.g., the ratio of monomer to crosslinker (M/C), the amount of PS and the molecular weight of macromolecular porogen was investigated. The surface properties of the monolithic polymer were characterized by scanning electron microscopy and nitrogen adsorption. The best polymerization condition was the M/C ratio of 7:3, and the PS concentration of 40 mg/ml. The poly(VPBA-co-EDMA) polymer was also applied to extract cis-diol flavonoid glycosides from the crude extraction of cotton flower. After treated by poly(VPBA-co-EDMA) for solid phase extraction, high purity ISO and HYP (>99.96%) can be obtained with recovery of 83.7% and 78.6%, respectively.

  19. Deconstructing Free Energies in the Law of Matching Water Affinities.

    PubMed

    Shi, Yu; Beck, Thomas

    2017-02-23

    The law of matching water affinities (LMWA) is explored in classical molecular dynamics simulations of several alkali halide ion pairs, spanning the size range from small kosmotropes to large chaotropes. The ion-ion potentials of mean force (PMFs) are computed using three methods: the local molecular field theory (LMFT), the weighted histogram analysis method (WHAM), and integration of the average force. All three methods produce the same total PMF for a given ion pair. In addition, LMFT-based partitioning into van der Waals and local and far-field electrostatic free energies and assessment of the enthalpic, entropic, and ion-water components yield insights into the origins of the observed free energy profiles in water. The results highlight the importance of local electrostatic interactions in determining the shape of the PMFs, while longer-ranged interactions enhance the overall ion-ion attraction, as expected in a dielectric continuum model. The association equilibrium constants are estimated from the smooth WHAM curves and compared to available experimental conductance data. By examining the variations in the average hydration numbers of ions with ion-ion distance, a correlation of the water structure in the hydration shells with the free energy features is found.

  20. Two measured completely different electron affinities for atomic Eu?

    NASA Astrophysics Data System (ADS)

    Msezane, A. Z.; Felfli, Z.

    2016-05-01

    Recently, the electron affinity (EA) of atomic Eu was measured to be 0.116?eV. This value is in outstanding agreement with the theoretically calculated values using the Regge pole and MCDF-RCI methods. Previously, the EA of Eu was measured to be 1.053 eV. In an attempt to resolve the discrepancy between the two measured values, we have adopted the complex angular momentum (CAM) method and investigated in the electron energy range 0.11 eV