DOI: 10.5061/dryad.jdfn2z3bd
title: Liquid chromatography tandem mass spectrometry of AMPA receptor containing vesicles

description: Regulated delivery of AMPA receptors (AMPARs) to the postsynaptic membrane
 is an essential step in synaptic strength modification, and in particular,
 long-term potentiation (LTP). While LTP has been extensively studied using
 electrophysiology and light microscopy, several questions regarding the
 molecular mechanisms of AMPAR delivery via trafficking vesicles remain
 outstanding, including the gross molecular make up of AMPAR trafficking
 organelles and identification and location of calcium sensors required for
 SNARE complex-dependent membrane fusion of such trafficking vesicles with
 the plasma membrane. Here, we isolated AMPAR containing vesicles (ACVs)
 from whole mouse brains via immunoisolation and characterized them using
 immunoelectron microscopy, immunoblotting, and liquid chromatography
 tandem mass spectrometry (LC-MS/MS). We identified several proteins on
 ACVs that were previously found to play a role in AMPAR trafficking,
 including synaptobrevin-2, Rabs, the SM protein Munc18-1, the
 calcium-sensor synaptotagmin-1, as well as several new candidates,
 including synaptophysin and synaptogyrin on ACV membranes. Here,
 we present three biological replicates of liquid chromatography tandem
 mass spectrometry of AMPA receptor containing vesicles.
description: <i>Liquid
 chromatography mass spectrometry</i> Purified ACVs were resuspended in
 50 ul 0.2 % Rapigest (Waters, Milford, MA) in 20 mM NH4HCO3 in 0.65 ml low
 protein binding polypropylene tubes before the addition of 5 mM DTT and
 incubation at 60˚C for 30 min. After this, iodoacetamide was added to a
 final concentration of 7.5 mM and samples were incubated for 30 additional
 minutes. Samples were then digested with 2.5 micrograms of sequencing
 grade trypsin (Trypsin Gold, Mass spectrometry grade, Promega, Madison,
 WI) at 37 °C, overnight. A second aliquot of trypsin (1.5 ug) was added,
 and the samples incubated for an additional 3 hours at 37 ˚C. After this,
 samples were acidified by adding 5% formic acid and incubated for 30
 minutes at room temperature. Tryptic peptides were recovered from the
 supernatant by C18 solid phase extraction using ZipTips (MilliporeSigma,
 Burlington, MA), eluted in two, 7 ul drops of 50% acetonitrile and 0.1%
 formic acid, and evaporated and resuspended in 5 ul 0.1% formic acid for
 LC-MS/MS analysis. Peptides resulting from
 trypsinization were analyzed on a QExactive Plus mass spectrometer
 (ThermoFisher Scientific) connected to a NanoAcquity™ Ultra Performance
 UPLC system (Waters). A 15-cm EasySpray C18 column (ThermoFisher
 Scientific) was used to resolve peptides (60-min 2–30% B gradient with
 0.1% formic acid in water as mobile phase A and 0.1% formic acid in
 acetonitrile as mobile phase B, at a flow rate of 300 nl/min). MS was
 operated in data-dependent mode to automatically switch between MS and
 MS/MS. MS spectra were acquired between 350 and 1500 m/z with a resolution
 of 70,000. For each MS spectrum, the top ten precursor ions with a charge
 state of 2+ or higher were fragmented by higher-energy collision
 dissociation. A dynamic exclusion window was applied which prevented the
 same m/z from being selected for 10 seconds after its
 acquisition. Peak
 lists were generated using PAVA in-house software (Guan et al., 2011). All
 generated peak lists were searched against the mouse subset of the
 UniProtKB database (SwissProt.2013.6.17) (plus the corresponding
 randomized sequences to calculate false discovery rate on the searches),
 using Protein Prospector (Clauser et al., 1999). The database search was
 performed with the following parameters: a mass tolerance of 20 ppm for
 precursor masses and 30 ppm for MS/MS, cysteine carbamidomethylation as a
 fixed modification, and acetylation of the N terminus of the protein,
 pyroglutamate formation from N terminal glutamine, and oxidation of
 methionine as variable modifications. A 1% false discovery rate was
 permitted at the protein and peptide level. All spectra identified as
 matches to peptides of a given protein were reported, and the number of
 spectra (peptide spectral matches, PSMs) was used for label free
 quantitation of protein abundance in the samples. Abundance index for each
 protein was calculated as the ratio of PSMs for a protein to the total
 PSMs for all components identified in the run divided by the polypeptide
 molecular weight.
description: 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.