Learning and memory result from synaptic plasticity - the ability of synaptic transmission to be modulated over time in response to stimuli. In contrast to the well-studied postsynaptic changes, presynaptic change in neurotransmitter release (exocytosis) from synaptic vesicles (SVs) is a mechanistically less understood form of synaptic plasticity. Exocytosis is regulated by phosphorylation, and the phospho-status of several exocytic proteins affects transmitter release. Exocytosis is followed by synaptic vesicle endocytosis which is controlled by proteins that are rapidly dephosphorylated upon nerve stimulation. We have used large-scale quantitative phosphoproteomics to study proteins from isolated nerve endings from rat brain (synaptosomes) that were at rest, depolarised for 10 sec. using KCl, or depolarised and allowed to recover for up to 15 min after depolarisation. After stimulation, the synaptosomes were lysed, subjected to protein precipitation and digested using trypsin. Phosphopeptides were enriched and fractionated using a combination of TiO₂, sequential elution from IMAC (SIMAC) and hydrophilic interaction liquid chromatography (HILIC) (collectively abbreviated TiSH). Peptides were analysed by LC-MS/MS on an LTQ-Orbitrap Velos/Elite and a 5600 TripleTOF instrument. The results show a prolonged, global reprogramming of the synaptic phosphoproteome, with an overrepresentation of exo- and endocytic proteins showing altered phosphorylation levels, and we therefore hypothesise that the initial short-term depolarisation changes the potential for subsequent neurotransmitter release on a much longer time-scale. Neurotransmitter release experiments are currently being performed and preliminary data seem to confirm the hypothesis – showing that the level of glutamate release from synaptosomes at a second stimulation 15 min after the initial one depends on the strength of the initial stimulation. In conclusion, our results indicate that depolarisation-induced phosphorylation changes in the pre-synaptic proteome constitute a previously undescribed form of pre-synaptic plasticity.