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Proteômica estrutural por espectrometria de massas : caracterização estrutural do complexo proteico FAK/Miosina ao nível molecular; Mass spectrometry based structural proteomics : structural characterization of FAK/Myosin complex at molecular level

Mariana Fioramonte
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 10/02/2012 PT
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Ligação cruzada acoplada à espectrometria de massas (MS) é uma técnica que permite a caracterização estrutural de proteínas e complexos proteicos, especialmente nos casos em que estes não são passíveis de serem analisados por técnicas de alta resolução. O experimento baseia-se na reação de uma proteína ou no complexo proteico com um reagente bifuncional (agente de ligação cruzada, ALC) seguido de análise proteômica shotgun por MS. Isso trás para a técnica todas as vantagens da MS, como alta sensibilidade, rapidez de análise e facilidade de uso. Somente resíduos de aminoácidos espacialmente próximos podem ser ligados covalentemente pelo ALC, de forma que os peptídeos contendo a ligação cruzada, identificadas por MS, servem como restrições de distância entre os aminoácidos na estrutura nativa da proteína ou complexo proteico. O conjunto de peptídeos contendo a ligação cruzada fornece, assim, um conjunto de restrições de distâncias entre os aminoácidos que pode ser utilizado para montar um modelo molecular da proteína ou do complexo. O objetivo principal deste trabalho foi a aplicação da técnica de ligação cruzada acoplada a espectrometria de massas no mapeamento da interação entre as proteínas quinase de adesão focal (FAK)...

Towards Functional Proteomics of Membrane Protein Complexes in Synechocystis sp. PCC 68031

Herranen, Mirkka; Battchikova, Natalia; Zhang, Pengpeng; Graf, Alexander; Sirpiö, Sari; Paakkarinen, Virpi; Aro, Eva-Mari
Fonte: The American Society for Plant Biologists Publicador: The American Society for Plant Biologists
Tipo: Artigo de Revista Científica
Publicado em /01/2004 EN
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45.86%
The composition and dynamics of membrane protein complexes were studied in the cyanobacterium Synechocystis sp. PCC 6803 by two-dimensional blue native/SDS-PAGE followed by matrix-assisted laser-desorption ionization time of flight mass spectrometry. Approximately 20 distinct membrane protein complexes could be resolved from photoautotrophically grown wild-type cells. Besides the protein complexes involved in linear photosynthetic electron flow and ATP synthesis (photosystem [PS] I, PSII, cytochrome b6f, and ATP synthase), four distinct complexes containing type I NAD(P)H dehydrogenase (NDH-1) subunits were identified, as well as several novel, still uncharacterized protein complexes. The dynamics of the protein complexes was studied by culturing the wild type and several mutant strains under various growth modes (photoautotrophic, mixotrophic, or photoheterotrophic) or in the presence of different concentrations of CO2, iron, or salt. The most distinct modulation observed in PSs occurred in iron-depleted conditions, which induced an accumulation of CP43′ protein associated with PSI trimers. The NDH-1 complexes, on the other hand, responded readily to changes in the CO2 concentration and the growth mode of the cells and represented an extremely dynamic group of membrane protein complexes. Our results give the first direct evidence...

Subunit architecture of multimeric complexes isolated directly from cells

Hernández, Helena; Dziembowski, Andrzej; Taverner, Thomas; Séraphin, Bertrand; Robinson, Carol V
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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Recent developments in purification strategies, together with mass spectrometry (MS)-based proteomics, have identified numerous in vivo protein complexes and suggest the existence of many others. Standard proteomics techniques are, however, unable to describe the overall stoichiometry, subunit interactions and organization of these assemblies, because many are heterogeneous, are present at relatively low cellular abundance and are frequently difficult to isolate. We combine two existing methodologies to tackle these challenges: tandem affinity purification to isolate sufficient quantities of highly pure native complexes, and MS of the intact assemblies and subcomplexes to determine their structural organization. We optimized our protocol with two protein assemblies from Saccharomyces cerevisiae (scavenger decapping and nuclear cap-binding complexes), establishing subunit stoichiometry and identifying substoichiometric binding. We then targeted the yeast exosome, a nuclease with ten different subunits, and found that by generating subcomplexes, a three-dimensional interaction map could be derived, demonstrating the utility of our approach for large, heterogeneous cellular complexes.

P177-T In Vivo Proteomics in Drosophila melanogaster by Tandem Affinity Purification of Protein Complexes

Rees, J. S.; Lowe, N.; Howard, J.; Hester, S.; St. Johnston, D.; Lilley, K.
Fonte: The Association of Biomolecular Resource Facilities Publicador: The Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /02/2007 EN
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55.83%
In order to characterize in vivo interactions in Drosophila melanogaster, and further our understanding of developmental processes, hybrid PiggyBac/P-element YFP traps with Strep Tag affinity tags were generated to isolate multi-protein complexes. The incorporated Strep affinity tag allows the target protein to be isolated from its native environment, by tandem affinity purification, along with any associating proteins. Purification efficiency can be followed visually by the YFP marker. Complex proteins are then identified by tandem mass-spectrometry against the Drosophila melanogaster database using the Mascot search engine.

EP7 Protein Microarrays

Brock, R.; Utz, P.
Fonte: The Association of Biomolecular Resource Facilities Publicador: The Association of Biomolecular Resource Facilities
Tipo: Artigo de Revista Científica
Publicado em /02/2007 EN
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35.74%
In the analysis of gene expression, DNA microarrays have found widespread application. However, expression on the mRNA level does not provide an accurate account of the functional state of a cell or tissue. First, mRNA levels do not reflect expression levels of proteins. Second, proteins are subject to a variety of post-translational modifications and enter into dynamically regulated molecular complexes. Because of their relative ease of handling, microarray-based approaches are gaining significance for the highly parallel analysis of proteins, complementing the more established proteomics approaches, such as mass spectrometry and gel electrophoresis. However, in comparison to DNA microarrays, protein microarrays are confronted with two major challenges: First, proteins possess very different physico-chemical characteristics, and activity depends on the preservation of their native conformation. Second, it is more difficult to identify a specific binder for a protein than to generate a highly specific DNA probe. To this point, protein array technology has focused on applications in auto-immunity, cancer, and microbiology. Many labs have collectively constructed arrays for studying three major classes of biomolecules: antibodies, cytokines and chemokines...

Two-dimensional Blue Native/SDS-PAGE Analysis Reveals Heat Shock Protein Chaperone Machinery Involved in Hepatitis B Virus Production in HepG2.2.15 Cells *S⃞

Liu, Kun; Qian, Lu; Wang, Jinglan; Li, Wenrui; Deng, Xinyu; Chen, Xilin; Sun, Wei; Wei, Handong; Qian, Xiaohong; Jiang, Ying; He, Fuchu
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em /03/2009 EN
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Hepatitis B virus (HBV) infection is a major health concern with more than two billion individuals currently infected worldwide. Despite the prevalence of infection, gaining a complete understanding of the molecular mechanisms of HBV infection has been difficult because HBV cannot infect common immortalized cell lines. HepG2.2.15, however, is a well established version of the HepG2 cell line that constitutively expresses HBV. Therefore, comparative proteomics analysis of HepG2.2.15 and HepG2 may provide valuable clues for understanding the HBV virus life cycle. In this study, two-dimensional blue native/SDS-PAGE was utilized to characterize different multiprotein complexes from whole cell lysates between HepG2.2.15 and HepG2. These results demonstrate that two unique protein complexes existed in HepG2.2.15 cells. When these complexes were excised from the gel and subjected to the second dimension separation and the proteins were sequenced by mass spectrometry, 20 non-redundant proteins were identified. Of these proteins, almost 20% corresponded to heat shock proteins, including HSP60, HSP70, and HSP90. Antibody-based supershift assays were used to verify the validity of the distinct protein complexes. Co-immunoprecipitation assays confirmed that HSP60...

Targeted tandem affinity purification of PSD-95 recovers core postsynaptic complexes and schizophrenia susceptibility proteins

Fernández, Esperanza; Collins, Mark O; Uren, Rachel T; Kopanitsa, Maksym V; Komiyama, Noboru H; Croning, Mike D R; Zografos, Lysimachos; Armstrong, J Douglas; Choudhary, Jyoti S; Grant, Seth G N
Fonte: Nature Publishing Group Publicador: Nature Publishing Group
Tipo: Artigo de Revista Científica
Publicado em 19/05/2009 EN
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35.85%
The molecular complexity of mammalian proteomes demands new methods for mapping the organization of multiprotein complexes. Here, we combine mouse genetics and proteomics to characterize synapse protein complexes and interaction networks. New tandem affinity purification (TAP) tags were fused to the carboxyl terminus of PSD-95 using gene targeting in mice. Homozygous mice showed no detectable abnormalities in PSD-95 expression, subcellular localization or synaptic electrophysiological function. Analysis of multiprotein complexes purified under native conditions by mass spectrometry defined known and new interactors: 118 proteins comprising crucial functional components of synapses, including glutamate receptors, K+ channels, scaffolding and signaling proteins, were recovered. Network clustering of protein interactions generated five connected clusters, with two clusters containing all the major ionotropic glutamate receptors and one cluster with voltage-dependent K+ channels. Annotation of clusters with human disease associations revealed that multiple disorders map to the network, with a significant correlation of schizophrenia within the glutamate receptor clusters. This targeted TAP tagging strategy is generally applicable to mammalian proteomics and systems biology approaches to disease.

Resolving mitochondrial protein complexes using non-gradient blue native polyacrylamide gel electrophoresis

Yan, Liang-Jun; Forster, Michael J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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45.86%
Blue native polyacrylamide gel electrophoresis (BN-PAGE) is a powerful technique for separation and proteomic analysis of high molecular weight protein complexes. It is often performed on gradient gels and is widely used for studying mitochondrial membrane complexes involved in electron transportation and oxidative phosphorylation. In this paper, we present an alternative BN-PAGE method that uses highly porous, non-gradient polyacrylamide gels for separation of rat brain mitochondrial protein complexes. Results demonstrate that this method not only resolves mitochondrial complexes I-V, allowing subsequent analysis by in-gel activity staining and mass spectrometry peptide sequencing, but also identifies Hsp60 polymers and dihydrolipoamide dehydrogenase (DLDH). Moreover, with this new method, it is shown for the first time that complex I and DLDH can be simultaneously detected on a single gel strip by in-gel activity staining. Overall, the method provides a simplified, non-gradient gel electrophoretic approach that should be useful in functional proteomics studies.

Dynamin-1 co-associates with native mouse brain BKCa channels: Proteomics analysis of synaptic protein complexes

Gorini, Giorgio; Ponomareva, Olga; Shores, Kevin S.; Person, Maria D.; Harris, R. Adron; Mayfield, R. Dayne
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
45.85%
In every synapse, a large number of proteins interact with other proteins in order to carry out signaling and transmission in the central nervous system. In this study, we used interaction proteomics to identify novel synaptic protein interactions in mouse cortical membranes under native conditions. Using immunoprecipitation, immunoblotting, and mass spectrometry, we identified a number of novel synaptic protein interactions involving soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), calcium-activated potassium channel (BKCa) alpha subunits, and dynamin-1. These novel interactions offer valuable insight into the protein-protein interaction network in intact synapses that could advance understanding of vesicle trafficking, release, and recycling.

Megadalton Complexes in the Chloroplast Stroma of Arabidopsis thaliana Characterized by Size Exclusion Chromatography, Mass Spectrometry, and Hierarchical Clustering*

Olinares, Paul Dominic B.; Ponnala, Lalit; van Wijk, Klaas J.
Fonte: The American Society for Biochemistry and Molecular Biology Publicador: The American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
EN
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46.06%
To characterize MDa-sized macromolecular chloroplast stroma protein assemblies and to extend coverage of the chloroplast stroma proteome, we fractionated soluble chloroplast stroma in the non-denatured state by size exclusion chromatography with a size separation range up to ∼5 MDa. To maximize protein complex stability and resolution of megadalton complexes, ionic strength and composition were optimized. Subsequent high accuracy tandem mass spectrometry analysis (LTQ-Orbitrap) identified 1081 proteins across the complete native mass range. Protein complexes and assembly states above 0.8 MDa were resolved using hierarchical clustering, and protein heat maps were generated from normalized protein spectral counts for each of the size exclusion chromatography fractions; this complemented previous analysis of stromal complexes up to 0.8 MDa (Peltier, J. B., Cai, Y., Sun, Q., Zabrouskov, V., Giacomelli, L., Rudella, A., Ytterberg, A. J., Rutschow, H., and van Wijk, K. J. (2006) The oligomeric stromal proteome of Arabidopsis thaliana chloroplasts. Mol. Cell. Proteomics 5, 114–133). This combined experimental and bioinformatics analyses resolved chloroplast ribosomes in different assembly and functional states (e.g. 30, 50, and 70 S)...

Proteomic Analysis Highlights the Molecular Complexities of Native Kv4 Channel Macromolecular Complexes

Marionneau, Céline; Townsend, R Reid; Nerbonne, Jeanne M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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35.89%
Voltage-gated K+ (Kv) channels are key determinants of membrane excitability in the nervous and cardiovascular systems, functioning to control resting membrane potentials, shape action potential waveforms and influence the responses to neurotransmitters and neurohormones. Consistent with this functional diversity, multiple types of Kv currents, with distinct biophysical properties and cellular/subcellular distributions, have been identified. Rapidly activating and inactivating Kv currents, typically referred to as IA (A-type) in neurons, for example, regulate repetitive firing rates, action potential back-propagation (into dendrites) and modulate synaptic responses. Currents with similar properties, referred to as Ito,f (fast transient outward), expressed in cardiomyocytes, control the early phase of myocardial action potential repolarization. A number of studies have demonstrated critical roles for pore-forming (α) subunits of the Kv4 subfamily in the generation of native neuronal IA and cardiac Ito,f channels. Studies in heterologous cells have also suggested important roles for a number of Kv channel accessory and regulatory proteins in the generation of functional IA and Ito,f channels. Quantitative mass spectrometry-based proteomic analysis is increasingly recognized as a rapid and...

Top-Down Mass Spectrometry of Supercharged Native Protein-Ligand Complexes

Yin, Sheng; Loo, Joseph A.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/03/2011 EN
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45.86%
Tandem mass spectrometry (MS/MS) of intact, noncovalently-bound protein-ligand complexes can yield structural information on the site of ligand binding. Fourier transform ion cyclotron resonance (FT-ICR) top-down MS of the 29 kDa carbonic anhydrase-zinc complex and adenylate kinase bound to adenosine triphosphate (ATP) with collisionally activated dissociation (CAD) and/or electron capture dissociation (ECD) generates product ions that retain the ligand and their identities are consistent with the solution phase structure. Increasing gas phase protein charging from electrospray ionization (ESI) by the addition of supercharging reagents, such as m-nitrobenzyl alcohol and sulfolane, to the protein analyte solution improves the capability of MS/MS to generate holo-product ions. Top-down proteomics for protein sequencing can be enhanced by increasing analyte charging.

Analysis of 953 Human Proteins from a Mitochondrial HEK293 Fraction by Complexome Profiling

Wessels, Hans J. C. T.; Vogel, Rutger O.; Lightowlers, Robert N.; Spelbrink, Johannes N.; Rodenburg, Richard J.; van den Heuvel, Lambert P.; van Gool, Alain J.; Gloerich, Jolein; Smeitink, Jan A. M.; Nijtmans, Leo G.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 23/07/2013 EN
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46.01%
Complexome profiling is a novel technique which uses shotgun proteomics to establish protein migration profiles from fractionated blue native electrophoresis gels. Here we present a dataset of blue native electrophoresis migration profiles for 953 proteins by complexome profiling. By analysis of mitochondrial ribosomal complexes we demonstrate its potential to verify putative protein-protein interactions identified by affinity purification – mass spectrometry studies. Protein complexes were extracted in their native state from a HEK293 mitochondrial fraction and separated by blue native gel electrophoresis. Gel lanes were cut into gel slices of even size and analyzed by shotgun proteomics. Subsequently, the acquired protein migration profiles were analyzed for co-migration via hierarchical cluster analysis. This dataset holds great promise as a comprehensive resource for de novo identification of protein-protein interactions or to underpin and prioritize candidate protein interactions from other studies. To demonstrate the potential use of our dataset we focussed on the mitochondrial translation machinery. Our results show that mitoribosomal complexes can be analyzed by blue native gel electrophoresis, as at least four distinct complexes. Analysis of these complexes confirmed that 24 proteins that had previously been reported to co-purify with mitoribosomes indeed co-migrated with subunits of the mitochondrial ribosome. Co-migration of several proteins involved in biogenesis of inner mitochondrial membrane complexes together with mitoribosomal complexes suggested the possibility of co-translational assembly in human cells. Our data also highlighted a putative ribonucleotide complex that potentially contains MRPL10...

A mass spectrometry-based hybrid method for structural modelling of protein complexes

Politis, Argyris; Stengel, Florian; Hall, Zoe; Hernández, Helena; Leitner, Alexander; Walzthoeni, Thomas; Robinson, Carol V.; Aebersold, Ruedi
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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35.8%
We describe a method that integrates data derived from different mass spectrometric (MS) techniques with a modelling strategy for structural characterization of protein assemblies. We encoded structural data derived from native MS, bottom-up proteomics, ion mobility-MS and chemical cross-linking MS into modelling restraints to compute the most likely structure of a protein assembly. We used the method to generate near-native models for three known structures and characterized an assembly intermediate of the proteasomal base.

Top Down Proteomics: Facts and Perspectives

Catherman, Adam D.; Skinner, Owen S.; Kelleher, Neil L.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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35.93%
The rise of the “Top Down” method in the field of mass spectrometry-based proteomics has ushered in a new age of promise and challenge for the characterization and identification of proteins. Injecting intact proteins into the mass spectrometer allows for better characterization of post-translational modifications and avoids several of the serious “inference” problems associated with peptide-based proteomics. However, successful implementation of a Top Down approach to endogenous or other biologically relevant samples often requires the use of one or more forms of separation prior to mass spectrometric analysis, which have only begun to mature for whole protein MS. Recent advances in instrumentation have been used in conjunction with new ion fragmentation using photons and electrons that allow for better (and often complete) protein characterization on cases simply not tractable even just a few years ago. Finally, the use of native electrospray mass spectrometry has shown great promise for the identification and characterization of whole protein complexes in the 100 kDa to 1 MDa regime, with prospects for complete compositional analysis for endogenous protein assemblies a viable goal over the coming few years.

Proteomic and metabolomic analyses of mitochondrial complex I-deficient mouse model generated by spontaneous B2 short interspersed nuclear element (SINE) insertion into NADH dehydrogenase (ubiquinone) Fe-S protein 4 (Ndufs4) gene

Leong, D.; Komen, J.; Hewitt, C.; Arnaud, E.; McKenzie, M.; Phipson, B.; Bahlo, M.; Laskowski, A.; Kinkel, S.; Davey, G.; Heath, W.; Voss, A.; Zahedi, R.; Pitt, J.; Chrast, R.; Sickmann, A.; Ryan, M.; Smyth, G.; Thorburn, D.; Scott, H.
Fonte: Amer Soc Biochemistry Molecular Biology Inc Publicador: Amer Soc Biochemistry Molecular Biology Inc
Tipo: Artigo de Revista Científica
Publicado em //2012 EN
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45.73%
Eukaryotic cells generate energy in the form of ATP, through a network of mitochondrial complexes and electron carriers known as the oxidative phosphorylation system. In mammals, mitochondrial complex I (CI) is the largest component of this system, comprising 45 different subunits encoded by mitochondrial and nuclear DNA. Humans diagnosed with mutations in the gene NDUFS4, encoding a nuclear DNA-encoded subunit of CI (NADH dehydrogenase ubiquinone Fe-S protein 4), typically suffer from Leigh syndrome, a neurodegenerative disease with onset in infancy or early childhood. Mitochondria from NDUFS4 patients usually lack detectable NDUFS4 protein and show a CI stability/assembly defect. Here, we describe a recessive mouse phenotype caused by the insertion of a transposable element into Ndufs4, identified by a novel combined linkage and expression analysis. Designated Ndufs4fky, the mutation leads to aberrant transcript splicing and absence of NDUFS4 protein in all tissues tested of homozygous mice. Physical and behavioral symptoms displayed by Ndufs4fky/fky mice include temporary fur loss, growth retardation, unsteady gait, and abnormal body posture when suspended by the tail. Analysis of CI in Ndufs4fky/fky mice using blue native PAGE revealed the presence of a faster migrating crippled complex. This crippled CI was shown to lack subunits of the “N assembly module”...

Proteomic Characterization of Plasma Membrane-proximal T Cell Activation Responses*

de Wet, Ben; Zech, Tobias; Salek, Mogjiborahman; Acuto, Oreste; Harder, Thomas
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
35.76%
Early downstream responses of T lymphocytes following T cell antigen receptor (TCR) activation are mediated by protein complexes that assemble in domains of the plasma membrane. Using stable isotope labeling with amino acids in cell culture and mass spectrometry, we quantitatively related the proteome of αCD3 immunoisolated native TCR signaling plasma membrane domains to that of control plasma membrane fragments not engaged in TCR signaling. Proteins were sorted according to their relative enrichment in isolated TCR signaling plasma membrane domains, identifying a complex protein network that is anchored in the vicinity of activated TCR. These networks harbor widespread mediators of plasma membrane-proximal T cell activities, including propagation, balancing, and attenuation of TCR signaling, immune synapse formation, as well as cytoskeletal arrangements relative to TCR activation clusters. These results highlight the unique potential of systematic characterizations of plasma membrane-proximal T cell activation proteome in the context of its native lipid bilayer platform.

A map of human protein interactions derived from co-expression of human mRNAs and their orthologs

Ramani, Arun K; Li, Zhihua; Hart, G Traver; Carlson, Mark W; Boutz, Daniel R; Marcotte, Edward M
Fonte: Nature Publishing Group Publicador: Nature Publishing Group
Tipo: Artigo de Revista Científica
Publicado em 15/04/2008 EN
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35.73%
The human protein interaction network will offer global insights into the molecular organization of cells and provide a framework for modeling human disease, but the network's large scale demands new approaches. We report a set of 7000 physical associations among human proteins inferred from indirect evidence: the comparison of human mRNA co-expression patterns with those of orthologous genes in five other eukaryotes, which we demonstrate identifies proteins in the same physical complexes. To evaluate the accuracy of the predicted physical associations, we apply quantitative mass spectrometry shotgun proteomics to measure elution profiles of 3013 human proteins during native biochemical fractionation, demonstrating systematically that putative interaction partners tend to co-sediment. We further validate uncharacterized proteins implicated by the associations in ribosome biogenesis, including WBSCR20C, associated with Williams–Beuren syndrome. This meta-analysis therefore exploits non-protein-based data, but successfully predicts associations, including 5589 novel human physical protein associations, with measured accuracies of 54±10%, comparable to direct large-scale interaction assays. The new associations' derivation from conserved in vivo phenomena argues strongly for their biological relevance.

Development and Applications of Chemical Labeling Protocols for Protein-Ligand Binding Analysis Using Bottom-Up Proteomics

Xu, Ying
Fonte: Universidade Duke Publicador: Universidade Duke
Tipo: Dissertação
Publicado em //2011
Relevância na Pesquisa
56.11%

Proteins fold into well-defined three-dimensional structures to carry out their biological functions which involve non-covalent interactions with other cellular molecules. Knowledge about the thermodynamic properties of proteins and protein-ligand complexes is essential for answering fundamental biological questions and drug or biomarker discovery. Recently, chemical labeling strategies have been combined with mass spectrometry methods to generate thermodynamic information about protein folding and ligand binding interactions. The work in this thesis is focused on the development and application of two such chemical labeling protocols coupled with mass spectrometry including one termed, SUPREX (stability of unpurified proteins from rates of H/D exchange), and one termed SPROX (stability of proteins from rates of oxidation). The work described in this thesis is divided into two parts. The first part involves the application of SUPREX to the thermodynamic analysis of a protein folding chaperone, Hsp33, and its interaction with unfolded protein substrates. The second part involves the development of a new chemical labeling protocol that can be used to make protein folding and ligand binding measurements on the proteomic scale.

In the first part of this work...

An informatic framework for decoding protein complexes by top-down mass spectrometry

Skinner, Owen S.; Havugimana, Pierre C.; Haverland, Nicole A.; Fornelli, Luca; Early, Bryan P.; Greer, Joseph B.; Fellers, Ryan T.; Durbin, Kenneth R.; Do Vale, Luis H. F.; Melani, Rafael D.; Seckler, Henrique S.; Nelp, Micah T.; Belov, Mikhail E.; Hornin
Fonte: Nature Publishing Group Publicador: Nature Publishing Group
Tipo: Relatório
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Efforts to map the human protein interactome have resulted in information about thousands of multi-protein assemblies housed in public repositories, but the molecular characterization and stoichiometry of their protein subunits remains largely unknown. Here, we report a computational search strategy for hierarchical top-down analysis, identification, and scoring of multi-proteoform complexes by native mass spectrometry.; The authors thank members of the Kelleher research group and Prof. V. Wysocki for helpful discussions and advice. O.S.S. is supported by a U. S. National Science Foundation Graduate Research Fellowship (2014171659). P.C.H. is a recipient of a Northwestern University's Chemistry of Life Processes Institute Postdoctoral Fellowship Award. L.H.F.D.V. is supported under CNPq research grant 202011/2012-7 from the Brazilian government. H.S.S is supported under the Science Without Borders scholarship 88888.075416/2013-00 from the Coordination for the Improvement of Higher Education Personnel, under the Brazilian government. This work was supported by grants from the W.M. Keck Foundation (DT061512) and the U.S. National Institutes of Health (GM067193) to N.L.K.