For post-translational protein modifications such as phosphorylation, the involved residues have been recently identified by mass spectrometers. However, it is not easy to efficiently detect the therapeutic target from the vast amount of proteomic data. Neuronal growth cones are specialized, highly motile structures formed at the tip of axons, that are indispensable for synaptogenesis in the developing brain and for neuronal plasticity in the adult brain. However, there is lack of information on the molecular basis of growth cones in the mammalian brain. We performed a phosphoproteomic analysis of growth cone membrane fractions (2 mg) isolated from the rat forebrain on postnatal day 1. We chose the more abundant peptides as research-targets, based on the hypothesis that abundant phosphorylated sites are involved in important biological functions. The phosphopeptides detected with high frequency at the 1st (Serine [S] 96) and 9th (Threonine [T] 172) positions were the phosphorylation sites of neuronal growth-associated protein - 43 kDa (GAP 43). C-jun N-terminal kinase (JNK) was responsible for phosphorylation at these sites, which increased during neuronal development and axonal regeneration. T172 phosphorylation was also confi rmed in rodents and primates. This review introduces our methodology for fi nding novel phospho-proteins as therapeutic molecular targets for specifi c diseases, along with their regulatory kinases. We believe that our serial approach, as illustrated here, can be applied to various research fi elds. In future research, we propose to demonstrate that pT172 antibody can be utilized as an axonal growth and regeneration marker in humans.
