We have adapted a luminex assay to quantitate tyrosine phosphorylation in tyrosine kinases. Active tyrosine kinases are also generally associated with increased overall tyrosine phosphorylation. We have collaborated with Dr. Todd Golub at the Broad Institute to capture more than 150 tyrosine kinases in a multiplex assay and detect the level of tyrosine phosphorylation in all these kinases using a pan-phosphotyrosine antibody (4G10). We used 150 human lung adenocarcinoma tumor tissue and corresponding adjacent normal lung tissue to profile tyrosine phosphorylation of these tyrosine kinases. We identified around 30 specific tyrosine kinases differentially phosphorylated in tumor tissue compared to adjacent normal lung. Examples of such potentially active tyrosine kinases are NTRK1, EPHA2, DDR. We are also performing a nanostring assay to measure transcript expression of these tyrosine kinases in a multiplex assay. In addition we have set-up collaboration with Dr. Paul Meltzer in the NCI intramural program to sequence around 1300 cancer-related genes, including the tyrosine kinases used in our luminex assay to identify specific mutations in a targeted manner.
