Synapses play important roles in neurotransmission and neuroplasticity. For an in-depth analysis of the synaptic proteome and phosphoproteome, synaptosomal proteins from whole mouse brain were analyzed by IEF and MS resulting in the largest synaptosome proteome described to date, with 2980 unique proteins identified with two or more peptides. At the same time, 118 synaptosomal phosphoproteins were identified, eight of which are reported for the first time as phosphorylated. Expression of selected proteins in synaptosomes was investigated by Western blot. We demonstrate that IEF is a powerful method to interrogate complex samples such as brain tissue both at the proteome and the phosphoproteome level without the need of additional enrichment for phosphoproteins. The detailed synaptoproteome data set reported here will help to elucidate the molecular complexity of the synapse and contribute to our understanding of synaptic systems biology in health and disease.