Proteome-Wide Quantitation by SILAC - LC-MS/MS in Proteomics: Methods and Applications - page 193

experiment with two labeling states; however, the SILAC
methodology can easily be expanded to three different labeling
states to compare simultaneously three different conditions ( see
Note 7 ). As an example we describe an experiment comparing
changes in protein expression levels in HeLa cells as a result
of 24 h stimulation with epidermal growth factor (EGF) ( see
Fig. 11.2 ) .

Fig. 11.2 Schematic overview of a typical SILAC-based quantitative proteomics experiment. Cells cultured in parallel are
labeled with different versions of the labeling amino acid(s) and mixed together. The combined cell lysate is fractionated
by, e.g., 1D gel electrophoresis and proteins are digested to peptides which are analysed by LC-MS/MS and identified by
database searching. The identified peptides are then quantified and used to calculate the protein ratio.

1. Prepare the SILAC labeling media as in Section 3.1.1 using
the optimal amino acids concentrations established from the
titration experiment described above.
2. From a dish with HeLa cells at 80-90% confluency aspirate
the media, rinse cells once with 3 mL PBS, and detach the
cells with 1 mL trypsin/EDTA solution. Transfer 300

3.2.1.MediaPreparation
andCellCulture

L
from the cell suspension into two dishes containing either
Lys0/Arg0 or Lys4/Arg6 media.
3. Passage the two cultures in parallel into the corresponding
SILAC media for five passages.
4. When 50% confluent at the fifth passage, supplement
the growth media of the Lys4/Arg6-labeled cells with
150 ng/mL EGF. Leave the cells grown in Lys0/Arg0
media untreated.
5. After 24 h of incubation wash the cell twice with 3 mL ice-
cold PBS.
6. Harvest cells as described in Steps 5-7 in Section 3.1.2 .

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