A new method for evaluation of intracellular protein kinase signals using mass spectrometry

Recently, comprehensive analysis in genome or proteome have attracted a lot of interest to many researchers in pharmacology, because of its useful information, such as expression profile of DNA, RNA and protein, to understand physiological events. However, it has not been possible to completely understand the cellular function using such information, because genes and proteins express their functions through extremely complicated interaction. On the other hand, total profile of the intracellular signals is expected to provide more detailed information to understand physiological events because various cellular functions are regulated directly by intracellular signals. We describe here an approach for the convenient and sensitive evaluation of intracellular protein kinase signals using mass spectroscopy. The method is based on a class of new peptide reagents and MALDI-TOF mass spectrometry. Using this system, activity changes in protein kinase A with a dosage of various pharmacological drugs into PC-12 cell were evaluated. These activity changes were found to have good correlation with the results of CREB-regulated gene expression, which was delivered into the cell line. We also evaluated the activity of protein kinase C and Src. This method can easily obtain the profile of many protein kinase activities and be useful for high throughput estimation of intracellular signalings, which is important to drug screening or evaluation of gene function.