Organelle Proteomics by Label-Free and SILAC-Based Protein Correlation Profiling - LC-MS/MS in Proteomics: Methods and Applications - page 258

2. PCP-SILAC : Mix the organelle-containing fractions from
the unlabeled cells to generate an internal standard.
Dilute the sucrose concentration by adding buffer ( see
Note 7 ), Fig. 15.2A .
3. PCP-SILAC : Combine the corresponding fractions from
the medium and heavy labeled preparations and dis-
tribute aliquots of the internal standard into these samples
( Fig. 15.2A ) .
4. PCP-SILAC : Gently invert to mix the internal standard
with the medium and heavy labeled fractions before pel-
leting the organelles by centrifugation. Remove the super-
natant after centrifugation. The samples can be stored at this
stage.

3.4.Gel
Electrophoresis
andLC-MSAnalysis

1. Dissolve the pellets in SDS sample buffer, heat the sam-
ples for 10 min at 75 ◦ C, reduce the disulfide bonds on cys-
teine in 10 mM DTT for 30 min followed by alkylation in
55 mM iodoacetamide for 20 min. Separate the proteins by
electrophoresis on NuPAGE R Bis-Tris 4-12% gradient gels
(Invitrogen) and stain the gels with Colloidal Blue to visual-
ize the proteins.
2. Cut the gel lanes into 10-15 slices. Transfer the slices to
Eppendorf tubes and cut them into small pieces using a
pointed scissor. Wash the gel pieces several times in 50 mM
ammonium bicarbonate and 50% acetonitrile and incubated
with 12.5 ng/ l trypsin in 50 mM ammonium bicarbon-

ate at 37 ◦ C overnight ( 15 ) . Extract the resulting peptides
with 1% TFA, desalt the samples on C18-STAGE tips ( see
Note 4 ), and elute the peptides into a 96-well plate. Remove
the organic solvent by vacuum centrifugation and redissolve
the samples in 1% TFA.
3. Inject the peptide mixtures onto a 75 ID reversed-phase
chromatography column filled with C18-resin. Elute the
peptides directly into the mass spectrometer with a 120 min
linear gradient of 95% buffer A to 50% buffer B.

μ

Fig. 15.2 (continued) standard for each fraction containing medium and heavy labeled proteins. Proteins in the com-
bined fractions are separated and digested with trypsin. ( B and C ) Selection of the organelle-containing fractions for the
PCP-SILAC experiment is determined by immunoblot analysis of an organelle marker protein or by peptide ion current
measurements as described in Fig. 15.1 using combined aliquots from the corresponding light, medium, and heavy
labeled fractions. ( D ) Relative enrichment profiles for each protein in the medium and heavy labeled organelle prepara-
tions are obtained by averaging the medium/light and heavy/light ratios for all peptides identified for the same protein
in each fraction. ( E and F ) Similar to PCP, the protein enrichment profile can be used to correctly assign organelle
proteins directly or by analyzing the distance between profiles for each protein and for a group of known organelle
proteins.