Organelle Proteomics by Label-Free and SILAC-Based Protein Correlation Profiling - LC-MS/MS in Proteomics: Methods and Applications - page 253

organelles are enriched but far from pure and the resulting list
of proteins obtained by mass spectrometry is crowded with unre-
lated proteins in the midst of genuine components. As a conse-
quence, the identified proteins provide corroborating rather than
unequivocal evidence of organelle association.
To get around this inadequacy it is important to distinguish
bona fide organelle components from co-purifying contaminants.
A successful strategy to achieve this goal has been the develop-
ment of quantitative profiling of organelles isolated by density
gradient centrifugation ( 3 - 5 ) . This strategy is based on algo-
rithms to compare the abundance profile of proteins in subcellular
fractions to the profiles of established organelle-associated pro-
teins. Deviation of each protein profile from the marker protein
profiles is a measure of its likelihood of being a genuine member
of the organelle. Key parameters for successful profiling experi-
ments are the reproducibility and the quality of organelle separa-
tion and the accuracy by which the abundance profile of proteins
is determined. Various quantitative methods based on mass spec-
trometry have been applied so far, including label-free quantita-
tion ( 6 ) , redundant peptide counting ( 7 ) , stable isotope labeling
by ICAT and iTRAQ ( 8 , 9 ) , and stable isotope labeling by amino
acid in cell culture (SILAC) ( 3 , 10 ) . These methods assign with a
high degree of confidence the core protein inventory of individ-
ual organelles but have also been demonstrated for more global
analyses of multiple cell organelles under different physiological
conditions ( 8 , 11 , 12 ) . In this chapter we describe protein cor-
relation profiling (PCP) in the format of label-free and SILAC-
based protein quantitation (PCP-SILAC).

2. Materials

2.1.CellCulture
andLysis

1. Standard culture medium: Dulbecco's Modified Eagle
Medium (DMEM) for PCP-SILAC custom culture medium
formulated identically to the standard medium but lacking
arginine and lysine.
2. Amino acids for PCP-SILAC ( 13 , 14 ) : Normal “light”
amino acids, L-lysine (Lys0) and L-arginine (Arg0)
hydrochloride; stable isotope-labeled “medium” amino
acids, L-lysine-4,4,5,5-d4 hydrochloride (Lys4) and
L-arginine- 13 C 6 hydrochloride (Arg6); and “heavy” amino
acids, L-arginine- 13 C 6 , 15 N 4 hydrochloride (Arg10) and
L-lysine- 13 C 6 , 15 N 2 hydrochloride (Lys8) (all Sigma-

Aldrich, Copenhagen, Denmark).
3. Supplements: Fetal bovine serum (FBS), in the case of PCP-
SILAC-dialyzed FBS (Gibco-Invitrogen, Carlsbad, CA);