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Combining RNA Interference Mutants and Comparative Proteomics to Identify Protein Components and Dependences in a Eukaryotic Flagellum

Eukaryotic flagella from organisms such as Trypanosoma brucei can be isolated and their protein components identified by mass spectrometry. Here we used a comparative approach utilizing two-dimensional difference gel electrophoresis and isobaric tags for relative and absolute quantitation to reveal...

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Autores principales: Portman, Neil, Lacomble, Sylvain, Thomas, Benjamin, McKean, Paul G., Gull, Keith
Formato: Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2645819/
https://www.ncbi.nlm.nih.gov/pubmed/19074134
http://dx.doi.org/10.1074/jbc.M808859200
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author Portman, Neil
Lacomble, Sylvain
Thomas, Benjamin
McKean, Paul G.
Gull, Keith
author_facet Portman, Neil
Lacomble, Sylvain
Thomas, Benjamin
McKean, Paul G.
Gull, Keith
author_sort Portman, Neil
collection PubMed
description Eukaryotic flagella from organisms such as Trypanosoma brucei can be isolated and their protein components identified by mass spectrometry. Here we used a comparative approach utilizing two-dimensional difference gel electrophoresis and isobaric tags for relative and absolute quantitation to reveal protein components of flagellar structures via ablation by inducible RNA interference mutation. By this approach we identified 20 novel components of the paraflagellar rod (PFR). Using epitope tagging we validated a subset of these as being present within the PFR by immunofluorescence. Bioinformatic analysis of the PFR cohort reveals a likely calcium/calmodulin regulatory/signaling linkage between some components. We extended the RNA interference mutant/comparative proteomic analysis to individual novel components of our PFR proteome, showing that the approach has the power to reveal dependences between subgroups within the cohort.
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spelling pubmed-26458192009-02-27 Combining RNA Interference Mutants and Comparative Proteomics to Identify Protein Components and Dependences in a Eukaryotic Flagellum Portman, Neil Lacomble, Sylvain Thomas, Benjamin McKean, Paul G. Gull, Keith J Biol Chem Genomics, Proteomics, and Bioinformatics Eukaryotic flagella from organisms such as Trypanosoma brucei can be isolated and their protein components identified by mass spectrometry. Here we used a comparative approach utilizing two-dimensional difference gel electrophoresis and isobaric tags for relative and absolute quantitation to reveal protein components of flagellar structures via ablation by inducible RNA interference mutation. By this approach we identified 20 novel components of the paraflagellar rod (PFR). Using epitope tagging we validated a subset of these as being present within the PFR by immunofluorescence. Bioinformatic analysis of the PFR cohort reveals a likely calcium/calmodulin regulatory/signaling linkage between some components. We extended the RNA interference mutant/comparative proteomic analysis to individual novel components of our PFR proteome, showing that the approach has the power to reveal dependences between subgroups within the cohort. American Society for Biochemistry and Molecular Biology 2009-02-27 /pmc/articles/PMC2645819/ /pubmed/19074134 http://dx.doi.org/10.1074/jbc.M808859200 Text en Copyright © 2009, The American Society for Biochemistry and Molecular Biology, Inc. Author's Choice Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) applies to Author Choice Articles
spellingShingle Genomics, Proteomics, and Bioinformatics
Portman, Neil
Lacomble, Sylvain
Thomas, Benjamin
McKean, Paul G.
Gull, Keith
Combining RNA Interference Mutants and Comparative Proteomics to Identify Protein Components and Dependences in a Eukaryotic Flagellum
title Combining RNA Interference Mutants and Comparative Proteomics to Identify Protein Components and Dependences in a Eukaryotic Flagellum
title_full Combining RNA Interference Mutants and Comparative Proteomics to Identify Protein Components and Dependences in a Eukaryotic Flagellum
title_fullStr Combining RNA Interference Mutants and Comparative Proteomics to Identify Protein Components and Dependences in a Eukaryotic Flagellum
title_full_unstemmed Combining RNA Interference Mutants and Comparative Proteomics to Identify Protein Components and Dependences in a Eukaryotic Flagellum
title_short Combining RNA Interference Mutants and Comparative Proteomics to Identify Protein Components and Dependences in a Eukaryotic Flagellum
title_sort combining rna interference mutants and comparative proteomics to identify protein components and dependences in a eukaryotic flagellum
topic Genomics, Proteomics, and Bioinformatics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2645819/
https://www.ncbi.nlm.nih.gov/pubmed/19074134
http://dx.doi.org/10.1074/jbc.M808859200
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