Combining enzymatic 18O-labeling and 2-D LC-MS/MS for a study of protein interactions in primary T cells

Abstract

Affinity-MS experiments with primary cells require alternative proteomic approaches, as the widely used metabolic labeling method SILAC is impracticable. We now describe a novel application of a recently developed 2-D LC-MS/MS approach for identification of phosphorylation-dependent protein interactions in human primary T cells. Using this approach, we identified a set of Tyr 595-phosphorylated ADAP interaction partners which belong to the larger TCR proximal signaling complex. The results show that a combination of two-dimensional RP–RP LC-MS/MS and ¹⁸O-labeling is a powerful means for peptide-based affinity MS experiments.