Fig. 1.

MIB-PRM workflow. Undigested protein lysate is passed through a gravity layered multidrug inhibitor beads column. Broad classes of activated kinases are preferentially enriched in the eluate. A tryptic digest of the eluate is loaded on a meter-scale monolithic silica-C18 column and resolved using a 6-h reverse phase chromatography gradient at a constant temperature of 40 °C. Online targeted detection of endogenous kinases by a highly multiplexed label-free PRM method provides high sensitivity and specificity of detection compatible with automated data processing.