Protein complexes have largely been studied by immuno-affinity purification and (mass spectrometric) analysis. While this approach has been widely and successfully used it is limited because it has difficulties reliably discriminating true from false protein complex components, identifying post-translational modifications and detecting quantitative changes in complex composition or state of modification of complex components. We have developed a protocol which enables us to determine, in a single LC-MS/MS analysis, the true protein constituents of a complex, to detect changes in the complex composition and to localize phosphorylation sites and estimate their respective stoichiometry. The method is based on the combination of four-plex iTRAQ isobaric labeling and protein phosphatase treatment of substrates. It was evaluated on model peptides and proteins and on the complex Ccl1-Kin28-Tfb3 isolated by tandem affinity purification from yeast cells. The two known phosphosites in Kin28 and Tfb3 could be reproducibly shown to be fully modified. The protocol was then applied to the analysis of samples immunopurified from Drosophila melanogaster cells expressing an epitope-tagged form of the insulin receptor substrate (IRS) homologue Chico. These experiments allowed us to identify 14-3-3e, 14-3-3 and the insulin receptor as specific Chico interactors. In a further experiment, we compared the immunopurified materials obtained from tagged-Chico-expressing cells that were either treated by insulin or left unstimulated. This analysis showed that hormone stimulation increases the association of 14-3-3 proteins with Chico and modulates several phosphorylation sites of the bait, some of which are located within predicted recognition motives of 14-3-3 proteins.