To stimulate phosphorylation and expression of the protein we focused on Jurkat T-cells, a human leukemic T cell lymphoblast cell line. Our first goal was to investigate whether the cells could be selectively stimulated to express phosphorylation at either Y492, Y493 or at both positions. This would allow us to probe the efficiency of the MIPs to enrich their targets in competition with excess of the other isoforms. For this purpose, we applied different bio-and chemical stimuli known to up-or down-regulate phosphorylation at the two sites and verified the outcome by Western Blot analysis using anti ZAP-70 phospho-antibodies. After profiling and identification of the overexpressed ZAP-70 phosphoproteins, whole cell lysate digestion or in-gel proteolytic digestion of the protein was performed, and the digest analyzed or used for further enrichment experiments (Fig. ).