Biology Reference
In-Depth Information
per the manufacturer's instructions. Wash the SpinColumn
with the 50
L ACN1 solution, centrifuge (1,000 ×
g
, 2 min),
and prime twice with 50
μ
L ACN2 solution, centrifuging
(1,000 ×
g
, 2 min) after each step.
4. Add peptide sample resuspended in ACN2 solution, centrifuge
(1,000 ×
g
, 2 min), and wash twice with 50
μ
L ACN2 solution,
centrifuging (1,000 ×
g
, 2 min) after each step. Finally elute with
25-50
μ
L ACN2 solution by centrifuging at 1,000 ×
g
for 2 min.
Concentrate and remove acetonitrile with a SpeedVac concen-
trator until 1-5
μ
5. Analyze peptide samples by liquid chromatography tandem
mass spectrometry (LC-MS/MS) using an automated data-
dependent acquisition method optimized for proteomic analy-
ses and characterizations.
6. Data produced by LC-MS/MS of samples can be analyzed
using software such as Mascot (Matrix Science, UK) to identify
proteins found in each fraction.
7. The proteins identifi ed in each individual fraction or pools of
multiple fractions can be compared to proteins associated with
published subcellular proteomes, most conveniently done
through the Arabidopsis SUBcellular database, SUBA [
10
].
In the example outlined in Fig.
2
we have used marker proteins
associated with particular organelles for which multiple experi-
mental assignations were available. Generally, only robustly
representative proteins should be included as organelle markers.
By monitoring the occurrence of these markers in individual
fractions, it is possible to track the relative migration of subcel-
lular compartments (
see
Note 22
).
8. Relative quantifi cation of proteins identifi ed in fractions
analyzed by LC-MS/MS can be achieved using specifi c
software, e.g., Scaffold 3. In Fig.
2
, spectral counts for those
proteins deemed to be robust representative markers of the
main organelles present in post-ZE-FFE fractions have been
aligned, thereby demonstrating subcellular compartment
separation.
μ
L of peptide solution remains in the tube.
Typical data produced and analyzed from FFE-separated endo-
membrane samples from Arabidopsis cell cultures is outlined in
Fig.
2
. These data demonstrate that the more electronegative Golgi
compartments (fractions 24 and under) can be almost completely
purifi ed from the ER and other contaminating compartments
using this technique. The parameters given here were selected for
ER-Golgi separation in a generic endomembrane-enriched sample.
Depending on the type of pre-FFE enrichment used (
see
Note 23
)
and specifi c adjustment of parameters, considerable scope exists for
separation or purifi cation of other compartments. Potential exists
3.5 Summary and
Future Uses of this
Technique
