Protein phosphorylation and dephosphorylation is a crucial fast signalling mechanism following depolarisation of presynaptic nerve terminals.  The strength of connections in neural circuits is adaptable, depends on brain activity and underlies learning and memory.  Depolarisation of axons and presynaptic terminals causes neurotransmitter filled synaptic vesicles to fuse with the membrane and release neurotransmitter into the synaptic cleft.  The the amount of neurotransmitter released can be regulated by a range of different biological processes directly and indirectly related to the cycle of synaptic vesicle fusion and biogenesis.  In the past, 32P metabolic labelling has provided a view of how presynaptic protein machinery engages with activity-dependent signalling.  We have used chemical depolarisation of isolated presynaptic nerve terminals to profile presynaptic signalling and compared this to 32P metabolic labelling.  Peptide derivatisation by iTRAQ or dimethylation with isotopes of different mass allowed quantification of phosphorylation levels over time.  We have shown that the type and level of stimulus determines the presynaptic response and have identified regulators of the synaptic vesicle cycle and other biological processes as targets for activity dependent phospho-signalling.