Organelle Proteomics by Label-Free and SILAC-Based Protein Correlation Profiling - LC-MS/MS in Proteomics: Methods and Applications - page 255

3. Methods

PCP and PCP-SILAC use the information inherent in the frac-
tionation profile of an organelle of interest to distinguish gen-
uine from co-purifying contaminants. The principle of the PCP
method is outlined in Fig. 15.1 . In this method, an organelle is
purified from tissue or cell lysates. Protein profiles are obtained
by integrating and normalizing peptide ion current signal inten-
sities extracted from LC-MS data. The PCP-SILAC method
( Fig. 15.2 ) requires SILAC-labeled proteins and has the advan-
tage that accurate protein profiles are obtained from isotope
ratios. The double PCP-SILAC experiment has the additional
advantage that protein profiles are obtained from two biological
replicates in a single experiment saving measuring time at the mass
spectrometer. The protocol described below is generic and does
not contain information related to the purification of a particu-
lar protein complex or cell organelle. We have successfully applied
PCP and PCP-SILAC to the characterization of human centro-
somes and autophagosomes which demonstrate the versatility of
the two methods. In the protocol presented below, we describe
in parallel PCP and PCP-SILAC with indications of steps specific
for each method.

Separate organelles
by density gradient

Collect
fractions

A

Separate proteins
Digest with trypsin
Quantitate by LCMS

B C

Peptide (organelle)
Peptide (contaminant)

Protein 1 (organelle)
Protein 2 (contaminant)

12

1

8

0.5

4

0

0

01234567 01234567

Fractions Fractions

Fig. 15.1 Protein correlation profiling. ( A ) Organelles are purified by density gradient centrifugation from cell lysate.
Proteins in each of the collected fractions are separated and digested with trypsin. The resulting peptides are measured
by liquid chromatography-mass spectrometry. ( B ) An abundance profile is obtained for each peptide by integrating the
peptide ion current in each fraction. ( C ) The profile for each peptide is normalized to the maximum signal and averaged
for all peptides identified for a given protein. Marker proteins for an organelle define a consensus profile and deviation of
each protein profile from the consensus profile is a measure of its likelihood of being a genuine member of the organelle.