g., Morciano et al., 2005). Indeed, Munc13, a component of the active zone, was only found in the docked synaptic vesicle fraction (Figure 3A). For further purification of docked synaptic vesicles, we carried out immunoisolation using nonporous microbeads covalently coated with antibodies against the synaptic vesicle protein synaptophysin. For comparison, a parallel immunoisolation step was carried out using the fraction
containing Dabrafenib free synaptic vesicles. Analysis of the immunoisolates for marker proteins revealed that the docked synaptic vesicle fraction contains both components of the active zone (Munc13) and of the presynaptic plasma membrane (Na+/K+-ATPase), both of which were absent from the free synaptic vesicle fraction and from immunoisolates using control IgG (Figure 3B). Mitochondrial proteins such as the succinate dehydrogenase complex subunit A (SDHA) that comigrated with docked synaptic vesicles on the gradient were not removed during immunoisolation raising the possibility that some mitochondria might be physically attached to the presynaptic plasma membrane and/or the active zone. To check for this possibility, aliquots of the gradient fractions enriched in docked synaptic vesicles were immobilized on coverslips and immunostained
for mitochondria selleck products and presynaptic markers. No significant overlap was detected (Figure S2). To identify proteins specifically associated with docking complexes, we carried out a quantitative proteomic comparison of the immunoisolated free and docked synaptic vesicle fraction using isobaric tag for relative and absolute quantitation (iTRAQ) (Ross et al., 2004) in combination with tandem mass spectrometry (LC-MS/MS). In this case, free and docked synaptic vesicle immunoisolates were digested by trypsin, followed by
labeling of the resulting peptides with isobaric tags of m/z 116 and m/z 117, respectively. These tags are chemically identical but give rise to reporter ions of different mass during fragmentation not in the MS/MS analysis, allowing for direct quantitative comparison. Both samples were combined after the labeling; the resulting peptides mixture prefractionated by SCX chromatography and analyzed by LC-MS/MS (see Figure 1B and Grønborg et al., 2010). A complete list of all proteins quantified/identified is shown in Table S1. In total, 493 proteins were identified from both fractions. As expected, a substantial portion (224 proteins) is of mitochondrial origin as classified by the MitoCarta and NCBI protein databases (Pagliarini et al., 2008). Of the remaining proteins, the largest fraction includes constituents of synaptic vesicles and proteins involved in exocytosis and recycling of synaptic vesicles (Figure 4).