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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...
Autores principales: | , , , , |
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Formato: | Texto |
Lenguaje: | English |
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American Society for Biochemistry and Molecular Biology
2009
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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. |
format | Text |
id | pubmed-2645819 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2009 |
publisher | American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
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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