Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 5 de 5
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Commun Biol ; 3(1): 735, 2020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-33277614

RESUMO

The TRAnsport Protein Particle (TRAPP) complexes act as Guanine nucleotide exchange factors (GEFs) for Rab GTPases, which are master regulators of membrane trafficking in eukaryotic cells. In metazoans, there are two large multi-protein TRAPP complexes: TRAPPII and TRAPPIII, with the TRAPPII complex able to activate both Rab1 and Rab11. Here we present detailed biochemical characterisation of Rab-GEF specificity of the human TRAPPII complex, and molecular insight into Rab binding. GEF assays of the TRAPPII complex against a panel of 20 different Rab GTPases revealed GEF activity on Rab43 and Rab19. Electron microscopy and chemical cross-linking revealed the architecture of mammalian TRAPPII. Hydrogen deuterium exchange MS showed that Rab1, Rab11 and Rab43 share a conserved binding interface. Clinical mutations in Rab11, and phosphomimics of Rab43, showed decreased TRAPPII GEF mediated exchange. Finally, we designed a Rab11 mutation that maintained TRAPPII-mediated GEF activity while decreasing activity of the Rab11-GEF SH3BP5, providing a tool to dissect Rab11 signalling. Overall, our results provide insight into the GTPase specificity of TRAPPII, and how clinical mutations disrupt this regulation.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Cromatografia Líquida , Humanos , Insetos , Modelos Moleculares , Conformação Proteica , Isoformas de Proteínas , Especificidade por Substrato , Espectrometria de Massas em Tandem , Proteínas rab de Ligação ao GTP/genética
2.
J Proteomics ; 225: 103844, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32480078

RESUMO

Structural Mass Spectrometry (SMS) provides a comprehensive toolbox for the analysis of protein structure and function. It offers multiple sources of structural information that are increasingly useful for integrative structural modeling of complex protein systems. As MS-based structural workflows scale to larger systems, consistent and coherent data interpretation resources are needed to better support modeling. Unlike the proteomics community, practitioners of SMS lack adequate computational tools. Here, we review new developments in the Mass Spec Studio: an expandable ecosystem of workflows for the analysis of complementary SMS techniques with linkages to modeling. Current functionality in the Studio (version 2) supports three major SMS workflows (crosslinking, hydrogen/deuterium exchange and covalent labelling) and two pipelines for structural modeling, with a special focus on data integration. The Mass Spec Studio is an architecture focused on rapid and robust extension of functionality by a community of developers. SIGNIFICANCE: This review surveys the new data analysis capabilities within the Mass Spec Studio, a rich framework for rapid software development specifically targeting the community of structural proteomics and structural mass spectrometry. Updates to crosslinking, hydrogen/deuterium-exchange and covalent labeling apps are provided as well as a utility for translating such analyses into restraints that support integrative structural modeling. These new capabilities, together with the underlying design tools and content, provide the community with a wealth of resources to tackle complex structural problem and design new approaches to data analysis.


Assuntos
Ecossistema , Proteínas , Espectrometria de Massas , Proteômica , Software
3.
Anal Biochem ; 586: 113416, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31499019

RESUMO

Microtubules (MTs) are key components in the cytoskeleton of the eukaryotic cell, and play roles in processes such as intracellular transport and cell division. An improved understanding MT regulation requires structural analysis of the extensive interactions between the MT lattice and its regulatory proteins, but MT interactions are challenging for even the most advanced structural methods to characterize. Integrative methods involving crosslinking mass spectrometry (XL-MS) can extend structural analysis to many interaction classes, but the representation of MTs in crosslinking data-sets has been surprisingly low. Here, we explore the basis for the underrepresentation of the MT lattice and present an enhanced method for mapping MT structural features using an optimized set of reagents, together with fluorescence detection to ensure MT structural integrity. Through the application of stringent identification criteria, 91 unique crosslinks were identified, 78 of which were uniquely matched to 7 distinct structural features of the MT lattice. Of note, 4 crosslinks were detected for the lattice-A protofilament organization. The lattice-A structure defines a "seam" or discontinuity in MTs and is an emerging site of interest for MT regulation. Our methodology should be broadly applicable to integrative structural studies involving any MT-protein interaction.


Assuntos
Reagentes para Ligações Cruzadas/química , Microtúbulos/química , Reagentes para Ligações Cruzadas/síntese química , Modelos Moleculares , Estrutura Molecular , Polimerização
4.
Mol Cell Proteomics ; 15(9): 3071-80, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27412762

RESUMO

The Mass Spec Studio package was designed to support the extraction of hydrogen-deuterium exchange and covalent labeling data for a range of mass spectrometry (MS)-based workflows, to integrate with restraint-driven protein modeling activities. In this report, we present an extension of the underlying Studio framework and provide a plug-in for crosslink (XL) detection. To accommodate flexibility in XL methods and applications, while maintaining efficient data processing, the plug-in employs a peptide library reduction strategy via a presearch of the tandem-MS data. We demonstrate that prescoring linear unmodified peptide tags using a probabilistic approach substantially reduces search space by requiring both crosslinked peptides to generate sparse data attributable to their linear forms. The method demonstrates highly sensitive crosslink peptide identification with a low false positive rate. Integration with a Haddock plug-in provides a resource that can combine multiple sources of data for protein modeling activities. We generated a structural model of porcine transferrin bound to TbpB, a membrane-bound receptor essential for iron acquisition in Actinobacillus pleuropneumoniae Using mutational data and crosslinking restraints, we confirm the mechanism by which TbpB recognizes the iron-loaded form of transferrin, and note the requirement for disparate sources of restraint data for accurate model construction. The software plugin is freely available at www.msstudio.ca.


Assuntos
Actinobacillus pleuropneumoniae/metabolismo , Reagentes para Ligações Cruzadas/química , Peptídeos/análise , Proteína B de Ligação a Transferrina/metabolismo , Transferrina/metabolismo , Actinobacillus pleuropneumoniae/química , Actinobacillus pleuropneumoniae/genética , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Modelos Moleculares , Mutação , Peptídeos/química , Ligação Proteica , Conformação Proteica , Software , Suínos , Espectrometria de Massas em Tandem , Transferrina/química , Proteína B de Ligação a Transferrina/química , Proteína B de Ligação a Transferrina/genética
5.
Rapid Commun Mass Spectrom ; 29(1): 119-27, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25462372

RESUMO

RATIONALE: Data analysis is a key step in mass spectrometry based untargeted metabolomics, starting with the generation of generic peak lists from raw liquid chromatography/mass spectrometry (LC/MS) data. Due to the use of various algorithms by different workflows, the results of different peak-picking strategies often differ widely. METHODS: Raw LC/HRMS data from two types of biological samples (bile and urine), as well as a standard mixture of 84 metabolites, were processed with four peak-picking softwares: Peakview®, Markerview™, MetabolitePilot™ and XCMS Online. The overlaps between the results of each peak-generating method were then investigated. To gauge the relevance of peak lists, a database search using the METLIN online database was performed to determine which features had accurate masses matching known metabolites as well as a secondary filtering based on MS/MS spectral matching. RESULTS: In this study, only a small proportion of all peaks (less than 10%) were common to all four software programs. Comparison of database searching results showed peaks found uniquely by one workflow have less chance of being found in the METLIN metabolomics database and are even less likely to be confirmed by MS/MS. CONCLUSIONS: It was shown that the performance of peak-generating workflows has a direct impact on untargeted metabolomics results. As it was demonstrated that the peaks found in more than one peak detection workflow have higher potential to be identified by accurate mass as well as MS/MS spectrum matching, it is suggested to use the overlap of different peak-picking workflows as preliminary peak lists for more rugged statistical analysis in global metabolomics investigations.


Assuntos
Cromatografia Líquida/métodos , Metabolômica/métodos , Software , Espectrometria de Massas em Tandem/métodos , Animais , Bile/química , Bases de Dados de Compostos Químicos , Cães , Metaboloma
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...