Abstract
Protein phosphorylation is a key post-translational modification that governs biological processes. Methods for monitoring the phosphorylation status of proteins are very important for the evaluation of diverse biological and pathological processes. Here we analyzed the site-specific phosphorylation of myelin basic protein (MBP) by protein kinase JNK1 (c-Jun N-terminal protein kinase) using liquid chromatography tandem mass spectrometry and iTRAQ reagent. Samples from time course reactions were differentially tagged using a multiplex reagent system, which enabled the simultaneous identification and relative quantification of phosphorylation. Three phosphorylation sites (Thr94, Thr97, and Ser164) were identified; all of them contained the JNK1 consensus sequence. The observed time course patterns can be classified into three different types corresponding to phosphorylated peptides, unphosphorylated peptides, and peptides with no phosphorylation sites. The method enables a better understanding of the complexities of kinase activity through the efficient analysis of time-dependent effects.
