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 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.
Portman
Neil
N
Sir William Dunn School of Pathology and Oxford Centre for Integrative Systems Biology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom.
Lacomble
Sylvain
S
Thomas
Benjamin
B
McKean
Paul G
PG
Gull
Keith
K
D000818:Animals; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D016054:DNA, Protozoan / Q000235:genetics / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005407:Flagella / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D040901:Proteomics; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D034622:RNA Interference; D016053:RNA, Protozoan / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism*
D016054:DNA, Protozoan; D015800:Protozoan Proteins; D016053:RNA, Protozoan; D034741:RNA, Small Interfering