Identification of Tyrosine Kinases and Their Substrates In Human Tumor Samples by 2D Gel Phosphotyrosine Western Blotting

Tyrosine kinases (TK) are known to initiate mitotic cell division and often drive cancer growth. Tyrosine kinase inhibitors (TKI), Gleevec for example, are a class of cancer drugs that sometimes have spectacular success at inhibiting cancer, but other times fail completely. High throughput genomic methods have been unsuccessful at predicting which of the many available TKIs match to which cancers.  One problem with genomic methods is that protein levels are often not proportional to mRNA levels. Pre (microRNA) and post-translation mechanisms exist at many levels in mammalian cells to regulate protein expression. Here we describe progress in direct identification of phospho-tyrosinylated proteins in human tumor tissue using the method of 2D gel phosphotyrosine Western blotting followed by mass spectrometry. Since tyrosine kinases often self-phosphorylate, the kinases themselves also light up. Flash-frozen lung, liver, kidney and pancreatic tumor samples along with normal tissue controls were purchased from a human tissue bank. The samples were homogenized and the proteins resolved by carrier ampholine 2D gel electrophoresis followed by western blotting with a PY20 anti-phosphotyrosine antibody. The ECL films were matched to duplicate Coomassie gels and spots cut from the latter for protein identification by mass spectrometry. Differences and similarities between the tumor sample western blot patterns will be discussed.