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Introduction: Protein phosphorylation is one of the most important post-translational modifications that regulate the function, localization, and binding specificity of target proteins. Specifically, protein phosphorylation works as a switch to turn “on” and “off” protein activity or a cellular pathway. Protein phosphorylation is also known to be both spatial and dynamic in its distribution. Therefore, a work-flow that provides the detailed information of phosphorylation site location, site occupancy under specific conditions, etc., is necessary. Current strategies such as ELISA, radio isotope workflow, and Western blotting lack the ability to ascertain independent information of each phosphorylation site. Our mass spectrometry–based method using a chemical labeling strategy provides dynamic information about phosphorylation on each phosphorylation site.
Method: A model system using JNK1 kinase and the substrate protein myelin basic protein (MBP) was selected. JNK1 phosphorylates serine and threonine residues just before the proline residue, typical of this MAP kinase family. The MBP substrate was incubated with JNK1 in vitro, and samples were removed at four different time points (0, 5, 30, and 60 min). These samples were digested and labeled with iTRAQ reagent in accordance with the standard procedure. A nano-LC-MS/MS experiment was performed using the 4000 Q TRAP System, and the data were analyzed by ProteinPilot Software.
Results: All of the peptides possessing a possible phosphorylation site motif in MBP were detected. For each identified peptide, the iTRAQ reagent reporter ions clearly showed the change in abundance of specific MBP peptides as they become phosphorylated by JNK1 over the time course of the assay. These results indicate that this workflow is a robust strategy for routinely characterizing the variation of phosphorylation through the interaction between protein substrate and kinase.