Mapping Protein–Protein Interactions by Quantitative Proteomics - LC-MS/MS in Proteomics: Methods and Applications - page 265

generation of mass spectrometers, contaminant proteins are also
often identified and carefully designed control experiments have
to be carried out to be able to eliminate these false-positive inter-
action partners from a data set. Quantitative experiments, either
via incorporated stable isotopes or via label-free quantitation, rep-
resent another possibility to address the question if proteins are
true binders or false-positive contaminants. Different isotope-
based labeling strategies exist ( 6 ) , one of them being stable iso-
tope labeling by amino acids in cell culture (SILAC) ( 7 ) where
cells are labeled metabolically. This strategy has been employed
successfully inter alia for the analysis of epidermal growth fac-
tor (EGF), platelet-derived growth factor (PDGF), and insulin-
dependent signaling complexes ( 8 - 11 ) . Chemical labeling strate-
gies via isotope-coded affinity tags (ICAT) ( 12 ) or via isobaric
tag for relative and absolute quantitation (iTRAQ) ( 13 ) have
also been successfully used for the analysis of protein complexes
( 14 , 15 ) . In this chapter we will present a detailed protocol for
the quantitative analysis of stimulus-specific signaling complexes
using SILAC-based liquid chromatography mass spectrometry
(LC-MS/MS), highlighting the possibility to distinguish weak
and strong interactions depending on the experimental setup.

2. Materials

2.1.CellCulture

1. Medium: Dulbecco's Modified Eagle Medium (DMEM)
deficient in lysine and arginine (Gibco-Invitrogen, Carlsbad,
CA).
2. Stable isotope-labeled “heavy” amino acids: L-arginine-
13 C 6 hydrochloride (Arg6) (Cambridge Isotope Labs,
Andover, MA; cat. no. CLM-2265), L-arginine- 13 C 6 , 15 N 4
hydrochloride (Arg10) (Sigma-Isotec, St. Louis, MO; cat.
no. 608033), L-lysine-4,4,5,5- d 4 hydrochloride (Lys4)
(Sigma-Isotec; cat. no. 616192), and L-lysine- 13 C 6 ,

15 N 2

hydrochloride (Lys8) (Sigma-Isotec; cat. no. 608041).
3. Supplements: Dialyzed fetal bovine serum (FBS); L-gluta-
mine (200 mM stock solution); penicillin/streptomycin
(10,000 U/10,000 g stock solution).

4. Additional: trypsin-EDTA solution (trypsin 200 mg/l and
versene-EDTA 500 mg/l); sterile phosphate-buffered saline
(PBS); filter units, MF75TM series (Nalge Nunc Interna-
tional, NY); recombinant EGF (PeproTech EC, London,
UK).
5. Cell lines:

μ