Biomedical Engineering Reference
In-Depth Information
Fig. 1
A comparative proteomic approach to identify new bilobe proteins. A schematic presentation of the
comparative proteomic approach. Each parasite cell contains a single copy each of the nucleus, the kinetoplast
(condensed mitochondrial DNA), the basal bodies that seed the single fl agellum, and the bilobe and the Golgi
that are located near the base of the fl agellum. Using different purifi cation protocols, samples containing fl a-
gellum only, or fl agellum in complex with basal bodies and bilobe, were prepared for comparative proteomic
analyses by iTRAQ. Proteins that were found more abundant in the fl agellar complex were deduced to be basal
bodies or bilobe proteins, which were further verifi ed and characterized experimentally
Apart from the centrins and Plk1, little was known about the
bilobe composition and functions. Biochemical purifi cation of the
bilobe has been diffi cult, partly due to its extensive association with
other cytoskeletal components [
6
]. We have therefore adopted two
different isolation and proteomic methods that allowed identifi ca-
tion of new bilobe and bilobe-associated proteins. In the fi rst
approach (Fig.
1
), different protocols were used to purify
T. brucei
fl agellum only or fl agellum in complex with basal bodies and
bilobe. Samples prepared using different protocols were then sub-
jected to comparative proteomic analyses by the isobaric tag for
relative and absolute quantitation (iTRAQ) method, which allowed
identifi cation of a new bilobe protein TbLRRP1 [
7
]. In the other
approach (Fig.
2
), YFP-TbLRRP1-labeled bilobe was immune iso-
lated for proteomic analyses, allowing further identifi cation of
proteins and structures, membranous or cytoskeletal, associated
with the bilobe [
8
]. Proteomic analyses, in combination with various
cell fractionation methods, are powerful approaches toward under-
standing of organelle compositions and functions.
