Protein phosphorylation is a typical posttranslational modification of protein, which plays important role in various metabolic regulation and signal transduction. Indeed, abnormal phosphorylation of key control protein has been detected in many cascades and is thought to underlie the associated cellular dysfunctions. Several signaling cascades have been implicated, affecting processes as varied as protein processing, protein expression, and subcellular protein localization, among others. Therefore proteomic profiling of phosphorylated proteins in clinical specimen is thought to be getting more important tool to detect abnormality in patient tissues. Two-dimensional gel electrophoresis-based proteomics is the most appropriate for analyzing protein phosphorylation because phosphorylated protein shows drift in isoelectric point in the first dimension isoelectric focusing. And phosphorylation is easily detected by various methods including autoradiography, immuno-blotting and in-gel staining with Pro-Q Diamond fluorescent dye. Phosphorylation can be confirmed by detecting neutral loss by MALDI-TOF MS in post-source decay (PSD) mode. We tried to profile phosphorylated protein human brain and human neuroblastoma cell line SH-SY5Y. Phosphoprotein specific staining with Pro-Q diamond was followed by SYPRO-Ruby staining and superimposed those two patterns in a Dual Color Display window of PDQuest . Both phosphorylated and unphosphorylated proteins were identified by paptide mass fingerprinting and phiosphorylation was confirmed. Thus obtained data of phosphorylated proteins in human brain tissue and human neuroblastoma SH-SY5Y cells.
