Fig. 1.

Experimental workflow for the analysis of tyrosine phosphorylated peptides via 5-plex SILAC. Breast cancer KPL-4 cells were grown in SM1–5 containing different combinations of unlabeled or stable isotopically labeled Lys, Arg, and Tyr. Each population was treated with the selective EGFR inhibitor erlotinib for the indicated times. SM1–5 cell lysates were combined and proteins were enzymatically digested. Tyrosine phosphorylated peptides were selectively enriched via two successive IPs with PY99- and 4G10-agarose conjugates. Eluted peptides were analyzed via nano-LC-MS/MS on an Orbitrap Velos.