Development of a 5-plex SILAC method tuned for the quantitation of tyrosine phosphorylation dynamics.

The propagation of phosphorylation downstream of receptor tyrosine kinases is a key dynamic cellular event involved in signal transduction, which is often deregulated in disease states such as cancer. Probing phosphorylation dynamics is therefore crucial for understanding receptor tyrosine kinases' function and finding ways to inhibit their effects. MS methods combined with metabolic labeling such as stable isotope labeling with amino acids in cell culture (SILAC) have already proven successful in deciphering temporal phosphotyrosine perturbations. However, they are limited in terms of multiplexing, and they also are time consuming, because several experiments need to be performed separately. Here, we introduce an innovative approach based on 5-plex SILAC that allows monitoring of phosphotyrosine signaling perturbations induced by a drug treatment in one single experiment. Using this new labeling strategy specifically tailored for phosphotyrosines, it was possible to generate the time profiles for 318 unique phosphopeptides belonging to 215 proteins from an erlotinib-treated breast cancer cell line model. Hierarchical clustering of the time profiles followed by pathway enrichment analysis highlighted epidermal growth factor receptor (EGFR or ErbB1) and ErbB2 signaling as the major pathways affected by erlotinib, thereby validating the method. Moreover, based on the similarity of its time profile to those of other proteins in the ErbB pathways, the phosphorylation at Tyr453 of protein FAM59A, a recently described adaptor of EGFR, was confirmed as tightly involved in the signaling cascade. The present investigation also demonstrates the remote effect of EGFR inhibition on ErbB3 phosphorylation sites such as Tyr1289 and Tyr1328, as well as a potential feedback effect on Tyr877 of ErbB2. Overall, the 5-plex SILAC is a straightforward approach that extends sample multiplexing and builds up the arsenal of methods for tyrosine phosphorylation dynamics.