Mapping Protein–Protein Interactions by Quantitative Proteomics - LC-MS/MS in Proteomics: Methods and Applications - page 269

Fig. 16.2: Example for analysis of stimulus-dependent dynamics of protein interactions using quantitative MS. ( A )Exper-
imental design. Cells are SILAC labeled using Arg0/Lys0, Arg6/Lys4, and Arg10/Lys8. Arg6/Lys4- and Arg10/Lys8-labeled
cells are stimulated with EGF for different time periods whereas Arg0/Lys0 cells are left untreated. Afterward cells are
mixed and lysed. EGFR and interacting proteins are purified by immuno-affinity enrichment using an anti-EGFR anti-
body. Precipitates are eluted in SDS loading buffer and separated by SDS-PAGE. The whole lane is cut in slices, proteins
are digested using trypsin, and resulting peptide mixtures are analyzed by LC-MS/MS. ( B ) Exemplified results. Peptides
derived from EGFR and stimulus-unspecific binders display ratios close to the original mixing 1:1:1 ratio. Stimulus-
specific binders show significantly different peptide ratios reflecting the degree of increased association with EGFR at
the corresponding time point. Thereby using triple encoding SILAC stimulus-dependent kinetics can be determined.

Lys0/Arg0 cells untreated to serve as control ( see Fig. 16.2 )
( see Note 4 ).
3. Remove medium from the culture dishes and scrape the
cells in ice-cold lysis buffer containing both protease and
phosphatase inhibitors. Incubate lysates on ice for 10 min.
Afterward remove cell debris by centrifugation. Save a small
aliquot (corresponding to approximately 30-50 gofpro-

tein) from each lysate and freeze these down ( see Note 5 ).

μ

1. Combine lysates and add 200 l protein A agarose to

pre-clear lysates of “sticky” proteins. Rotate for minimally
30 min at 4 ◦ C( see Note 6 ).
2. Remove protein A agarose by passing lysate through a dis-
posable Poly-Prep column and save a small aliquot corre-
sponding to approximately 30-50

μ

3.3.
Immunoprecipitation
andGel
Electrophoresis

μ gofprotein( see Note

7 ).
3. Add immobilized anti-EGFR antibody into the mixed lysates
and rotate for 4-6 h at 4 ◦ C( see Note 8 ).
4. Wash the beads three times with five volumes of ice-cold lysis
buffer. Resuspend the beads in SDS loading buffer and elute
precipitated complexes by heating at 75 ◦ C for 10 min.