Research output: Contribution to Journal/Magazine › Journal article › peer-review
Research output: Contribution to Journal/Magazine › Journal article › peer-review
}
TY - JOUR
T1 - Combining RNA interference mutants and comparative proteomics to identify protein component interactions and dependencies in a eukaryotic flagellum.
AU - Portman, Neil
AU - Lacomble, Sylvain
AU - Thomas, Benjamin
AU - McKean, Paul G.
AU - Gull, Keith
PY - 2009/2
Y1 - 2009/2
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=65549110811&partnerID=8YFLogxK
U2 - 10.1074/jbc.M808859200
DO - 10.1074/jbc.M808859200
M3 - Journal article
VL - 284
SP - 5610
EP - 5619
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 1083-351X
IS - 9
ER -