Organelle Proteomics by Label-Free and SILAC-Based Protein Correlation Profiling - LC-MS/MS in Proteomics: Methods and Applications - page 256

3.1.Organelle
Fractionation

1. PCP : Prepare lysates from cells or tissues ( see Note 1 )and
isolate the organelle of interest by density gradient centrifu-
gation ( see Note 2 , Fig. 15.1A ) .
2. PCP-SILAC : Prepare lysates from three SILAC-labeled cell
populations (light, medium, and heavy) and isolate in par-
allel the organelle of interest by density gradient centrifuga-
tion ( see Note 3 , Fig. 15.2A ) .
3. Elute the gradients by making a hole in the bottom of the
tube with a hot 20 G needle and collect, e.g., 0.5 mL frac-
tions from the bottom of the tube into 2 mL Eppendorf
tubes. In the case of gradients of low density, collect fractions
from the top or the bottom of the gradient by pipetting or
by the use of a gradient collector.

3.2.Determinationof
Organelle-Containing
Fractions

1. Withdraw aliquots (e.g., 50 L) from each fraction. Deter-

mine the distribution of organelle proteins by LC-MS or by
the use of organelle-specific antibodies for immunoblot anal-
ysis or for immunofluorescent microscopy of organelles sed-
imented onto a coverslip.
2. PCP-SILAC : Combine the 50

μ

L aliquots of the corre-
sponding fractions for each of the three organelle prepara-
tions (light, medium, and heavy) to reduce the number of
samples to be tested by LC-MS ( Fig. 15.2B ) .
3. Digest the samples in-solution ( see Note 4 ), purify the
resulting peptide on STAGE tips ( see Note 5 ), and subject
the samples to LC-MS analysis and peptide identification as
described below.
4. PCP : Quantify the abundance of each peptide in each frac-
tion by the integration of the ion current signal intensity
extracted from the precursor ion mass spectra.
5. PCP-SILAC : Quantify the abundance of each peptide in
each fraction by the integration of the ion current signal
intensity extracted from the precursor ion mass spectra for
each of the three signals corresponding to the light, medium,
and heavy isotope clusters.
6. Plot the peptide abundance profiles ( Figs. 15.1B and
15.2C ) to determine the distribution of organelle proteins.
7. PCP : Dilute the sucrose concentration by adding buffer and
pellet the organelles by centrifugation. Remove the super-
natant after centrifugation. The samples can be stored at this
stage.

μ

3.3.Generationof the
InternalStandard
andCombinationof
Fractions

1. PCP-SILAC : Select organelle-containing fractions, e.g., two
to three fractions left and right to the peak fraction ( see
Note 6 ).