Fig. 1.

Overview of the label-free quantitative immuno-competitive capture mass spectrometry (ICC-MS) experimental procedure for the detection of protein interactors. Cell lysates are first pre-incubated with increasing concentrations of free antibody and then individually immunoprecipitated using the same antibody immobilized on agarose beads. After discarding the unbound fractions and performing several washing steps, the eluted fractions are separated by SDS-PAGE, proteins digested with trypsin, and extracted peptides analyzed by LC-MS/MS on an Orbitrap. Proteins are quantified on the basis of their corresponding peptide EICs and protein signals are subjected to a statistical analysis in order to derive the concentration-dependent signal decrease of the specific interactors.