(34a) Identification of Activated Receptor Tyrosine Kinases Using 2DE Western Blot Image Overlays

Receptor tyrosine kinases (RTK) are a class of trans-membrane protein activated by phosphorylation of tyrosine groups on the cytosolic chain domain. Activated RTKs, often growth factors like EGFR (epidermal growth factor receptor), are known drivers of cancer growth. Several RTK inhibitors have been tested in clinical trials and are approved by the FDA for cancer treatment. However, physicians have difficulty matching patients to the correct RTK inhibitor during the critical period of early treatment. Cancer growth triggered by one kind of RTK does not respond to an inhibitor of a different kind. Other oncogenic proteins, RAS for example, trigger cancer growth by different pathways and also do not respond to RTK inhibitors.  Tests for inactive RTKs, immunohistochemistry for example, do not correlate with inhibitor success. A test for detection and identification of specific, activated RTKs in tumor tissue is needed.

 We have previously shown that tyrosine phosphorylation (activation) of putative RTKs can be detected in human tumors using carrier ampholine 2-dimensional gel electrophoresis (CA-2DE) in conjunction with Western blotting (WB).  Initial attempts using mass spectrometry to identify activated RTKs detected by WB were unsuccessful. Attempts to identify the activated RTKs independently using protein arrays were also unsuccessful. RTKs are high molecular weight, membrane proteins that do not dissolve without the solubilizing agent, sodium dodecyl sulfate (SDS). Protein arrays are incompatible with SDS;  CA-2DE is compatible with this detergent.  The very high sensitivity of the WB method is due to the high affinity of the PY20 antibody for the phosphotyrosine epitope.

The approach taken in this work is to identify the activated RTKs by duplex 2D WB. First, CA-2DE WB with the anti-phosphotyrosine antibody is performed and images obtained. The blot is stripped, and then reprobed with an antibody against a known RTK such as EGFR. Because both WB images are obtained from the same membrane, they are superimposable and can be easily overlaid using software called SameSpots. Exact overlay of the phosphotyrosine WB (activation pattern) with the anti-RTK protein WB (identity pattern) confirms identification of the activated RTK. Mismatches are easy to detect. Successful results from analysis of a set of six human lung cancer tumors obtained from a tissue bank are discussed.