The primary goal of the project is to develop mass spectrometry (MS) based assays capable of globally quantifying changes in kinase-specific phosphorylation stoichiometry. The specific work related to this proposed research includes a) quantitatively measuring the change of substrate phosphorylation stoichiometry corresponding to the change of the specific kinase activity in a complex biological system using high resolution liquid chromatography mass spectrometry (LC-MS) and b) quantifying the abundance change for selected kinase specific substrate candidate biomarkers after inhibitor/drug treatment using high sensitivity and specificity LC triple quadrupole MS for targeted sample analysis in which MS will be operated in a highly selective multiple ion monitoring (MRM) mode. Due to the extremely high sensitivity and broad dynamic range requirements for all the LC-MS measurements in both tasks a) and b) a suite of LC-MS technologies developed at Pacific Northwest National Laboratory (PNNL) including high resolution capillary LC, high efficiency electrospray ionization (ESI) source and unique ion funnel based MS interface will be used to integrate with the commercial high resolution Orbitrap MS and triple quadrupole MS to gain several orders of magnitude sensitivity improvement as compared to the commercially available MS instruments.