An automated online immobilized metal affinity chromatography/high-performance liquid chromatography mass spectrometric (IMAC-HPLC/MS/MS) method was developed to study cytidine 3',5'-cyclic monophosphate (cCMP)-specific protein phosphorylation, analogous to a previously successful offline IMAC method using microvolume IMAC pipette tips. The optimized method identified murine brain phosphoproteins selectively modified by challenge with cCMP, using manual interpretation of the results to confirm both phosphorylation and selectivity of response to cCMP. A number of proteins identified by this strategy have potential roles in hyperproliferation, a previously reported response to elevated levels of cCMP.
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