(The FASEB Journal. 2008;22:1054.2.)
© 2008 FASEB
Profiling Kinase Pathway Activation and Inhibitor Activity Using a Novel Antibody Array
Jeffrey Alan Radding,
Christine Rauh-Adelmann,
Amy B. Hall,
Cheryl Murphy,
Lucy G. Yen,
James R. Graham,
Timothy K. Nadler and
Neal F. Gordon
Epitome Biosystems, Waltham, MA
ABSTRACT
Broad screening of protein phosphorylation has wide-ranging utility for understanding the impact of disease or drug treatment on cell signaling pathways. To address this need, we have developed an antibody array for measuring tyrosine signaling across 45 phosphotyrosine sites across multiple signaling pathways (Ti-Tyr TM Profiling Chip). The approach utilizes a novel strategy of measuring peptide fragments liberated by protease digestion of the sample prior to analysis, and allows for both multi-site profiling and absolute quantification using synthetic peptide standards. The utility of the Ti-TyrTM chip is demonstrated by measuring specific modulation of pathway phosphorylation in stimulated cells in the presence and absence of distinct kinase inhibitors. Phosphorylation changes detected in chip targets correlate with known biological effects using comparable conditions. The use of peptide standards on the chip allows for an absolute quantitative assessment of the same stimulation conditions or inhibitor treatments across different cell lines, for example EGF stimulation in A431 and PC-3 cancer lines. The Ti-Tyr TM Tyrosine Chip should improve the ability to understand the effects of cell stimuli, the underlying basis of disease and the impact of drug treatments across multiple cell pathways and cell types.
