Mapping Protein–Protein Interactions by Quantitative Proteomics - LC-MS/MS in Proteomics: Methods and Applications - page 268

Table 16.1
Examples of different SILAC-based approaches to study
protein-protein interactions a

Type of
interaction

Type of affinity
enrichment

References

Protein-protein Using recombinant

domain as a bait

( 8 , 29 )

Protein-protein Immunoprecipitation

of overexpressed
tagged protein

( 25 , 26 , 29 - 32 )

Protein-protein Immunoprecipitation

of endogenous
complexes

( 9 - 11 , 17 , 28 , 33 ,
34 )

Sequence-directed
Protein-peptide

Using synthetic
peptides as a bait:
mutated versus
control

( 35 )

PTM-directed
Protein-peptide

Using modified
synthetic peptides
as a bait

( 11 , 34 , 36 - 39 )

Protein-DNA Synthetic DNA

oligonucleotides
(mutated versus
control)

( 40 )

a For a recent review on the topic see (41).

The EGFR signaling network has been extensively studied by our
group resulting in identification of several novel components of
the signaling cascade and delineation of the site-specific phospho-
rylation kinetics of many novel phosphorylation sites ( 8 , 9 , 17 ,
18 ) ( see Note 2 ). However, the experimental approach provided
below is generic and the described protocol can be used as a gen-
eral outline for the quantitative analysis of stimulus-dependent
protein interactions.

3.1.CellCulture

Cells should be grown for at least five cell doublings in SILAC
medium to ensure full incorporation of the labeled amino acids
( 19 ) ( see Note 3 ). We recommend to use four 15 cm dishes per
condition (approximately 4-5

10 7 cells). ×

3.2.Treatment
ofCellsandCell
Lysis

1. When cells are approximately 70% confluent, replace
medium with the respective serum-free SILAC medium. For
adherent HeLa cells, 12-14 h of serum deprivation is an
optimal time frame.
2. Stimulate Lys4/Arg6- and Lys8/Arg10-labeled cells for a
different length of time with 150 ng/ml EGF,

leaving