PNNL

Abstract

Identification of tyrosine phosphorylated proteins by traditional SDS-PAGE requires a priori assumptions about the identity of proteins likely to be phosphorlated under the experimental conditions used, as well as the availability of a specific antibody to that protein. We have used a mass spectrometry-based approach to avoid these limitations. Cell lysates obtained from wild-type and SRC-transformed fibroblasts were immunoprecipitated with the anti-phosphotyrosine antibody 4G10 and fractionated by one dimensional SDS-PAGE. Bands were excised, digested with tyrosine, and the extracted peptides subjected to MALDI-TOF analysis. Proteins identified as tyrosine phosphorylated in SRC-transformed cells include IZ-RasGAP, GAP-associated p190, and blk, a member of the src kinase super-family. We next investigated the ability of carboxyamidotriazole (CAI), a novel chemotherapeutic agent currently in Phase II clinical trials for prostate cancer and head and neck cancers, to modulate the pattern of tyrosine phosphorylated proteins in SRC transformed cells and in the SKOV3 ovarian tumor cell line. Treatment of with CAI at 1 uM resulted in loss of tyrosine phosphorylation in SRC-transformed cells, but not in the ovarian tumor cells. These results indicate that MADI-MS analysis of immunoprecipitated proteins separated on one dimensional SDS-Page gels is an effective strategy for identifying changes in protein modification or association as a result of experimental or therapeutic treatment. This approach, coupled with cDNA microarray data on mRNAs up-regulated in ovarian tumor cells, can be used to monitor the effectiveness of novel chemotherapeutics on functionally important proteins.