Updated project metadata. Cultured hippocampal neurons from mice were treated with high concentration KCl for five minutes to depolarise the plasma membrane and model sustained brain activity. A high KCl condition was compared to low KCl using phosphoproteomics. Phosphopeptides were enriched from trypsin digested bio-replicates of the high and low KCl conditions and analysed by mass spectrometry. Approximately 20,000 phosphopeptides were identified enabling a comparison of the relative level of phosphorylation in response to depolarisation.