Liquid chromatography tandem mass spectrometry of AMPA receptor containing vesicles

No additional information is needed to use this dataset. To replicate this dataset, one would need to purify AMPA receptor containing vesicles (see below).

To isolate ACVs, we followed a previously developed protocol for synaptosome generation and synaptic vesicle isolation (Ahmed et al., 2013) and extensively modified it to specifically purify ACVs. 8-12 ~P20 CD-1 mice were sacrificed using an isoflurane chamber, and whole brains were immediately removed and homogenized. (See Figure 1 for full summary.) This initial homogenate was spun in a JA-20 rotor at 2700 RPM (880 G) for 10 minutes to pellet blood vessels and other large cellular debris. The supernatant was then spun at 10,000 RPM (12,064 G) for 15 minutes to pellet synaptosomes. The supernatant was discarded and the periphery of the pellet was resuspended, which helps to remove mitochondria, before spinning at 11,000 RPM (14,597 G) for 15 minutes. The supernatant was again discarded, and the pellet resuspended to 5 ml total volume. The suspension was added to a Dounce homogenizer along with 45 ml of ultrapure water and was briefly homogenized to hypoosmotically lyse the synaptosomes. Immediately afterwards, 60 ul of 1 mg/ml pepstatin A and 120 ul of 200 mM PMSF in 1M HEPES was added. This solution was spun at 19,500 RPM (45,871 G) for 20 minutes to pellet plasma membrane and large cellular debris while leaving small organelles like vesicles in solution. The supernatant was then removed and spun in a Ti-70 ultracentrifuge at 50,000 RPM (256,631 G) for 2 hours at 4 °C  to pellet small organelles like trafficking vesicles. The pellet was transferred to a small homogenizer and resuspended in 2 ml of PBS by homogenization and mechanically sheared through a 27-gauge needle. The concentration of LP2 was determined using BCA and aliquoted into 2 mg aliquots at approximately 5 µg/µl. Any LP2 not used immediately for ACV isolation was flash frozen with liquid nitrogen and stored at -80 °C until use.

To isolate ACVs from LP2, 1 aliquot of 2 mg LP2 was diluted to 1 ml total volume in 0.5% BSA in PBS, 5 ul of mouse anti-GluA1 monoclonal antibody (1ug/µl, Synaptic Systems, Gottingen, Germany) was added and allowed to bind while rotating for 12 hours at 4 °C. To prevent nonspecific binding, 50 µl of paramagnetic protein G beads (Dynabeads, ThermoFisher Scientific, Waltham, MA) were washed 3 times in 0.5% BSA in PBS for 15 minutes on ice and then 3 times in PBS for 5-minute washes on ice prior to addition of LP2. The LP2 mixture was then added to the beads and rotated for 2 hours at 4 °C. Dynabeads were separated from solution using a magnet, and the flow through was collected for Western blot analysis. ACVs were then gently eluted with three, 20-minute washes with 33 ul of GluA1 peptide (20 ug/ul) representing the same synthetic peptide the antibody was created against (sequence: SHSSGMPLGATGL) (GenScript Biotech, Piscataway, NJ). ACVs were then immediately used and continually stored on ice at 4 °C. Protein concentration was measured by Bradford assay. Serial dilutions of BSA were used to generate a standard curve.