Biotinylation by antibody recognition

Collect the brain sections at 240-micron intervals across the neuroaxis, place them into a net well (Brain research laboratories) and wash 3 times for 1h 0m 0s each in TBST.

Place the sections in 0.3% hydrogen peroxide and 0.1% sodium azide diluted in blocking buffer for 1h 0m 0s at Room temperature to quench endogenous peroxidases.

Rinse the sections briefly in TBST and incubate in anti-PSER129 antibody EP1536Y diluted 1:50,000 in blocking buffer 1h 0m 0s at 4°Cwith gentle agitation.

The following day, wash the sections 3 times in TBST, then incubate with biotinylated anti-rabbit antibody diluted 1:200 in blocking buffer for 1h 0m 0s at Room temperature

Wash the sections 3 times in TBST, incubate with ABC reagent for 1h 0m 0s, and wash off with borate buffer.

Incubate the sections with borate buffer containing biotinyl tyramide as described above.

Wash the sections 1h 0m 0s with TBST, gather in a 1.5mL Eppendorf tube, 3000x g to pellet floating sections, and discard the supernatant.

Briefly sonicate each sample in 1mL of crosslink reversal buffer (refer materials section) and heat for 0h 30m 0s at 98°C followed by 1h 0m 0s at 90°C.

Centrifuge the sample 20000x g of the samples and then dilute the supernatant 1:10 in modified TBST (refer materials section).

Incubate each sample with 40mg of streptavidin magnetic beads (Thermofisher Scientific) for 2h 0m 0s at Room temperature with constant mixing.

Collect the beads using a magnetic stand (Thermofisher Scientific), wash the beads 3 times in modified TBST, and then 2h 0m 0s in 10mL of stringent wash buffer (refer materials section).

The following day, collect the beads using magnetic stand and resuspend in 100µL 1 X Bolt LDS sample buffer with reducing agent (Thermofisher) then heat for 0h 10m 0s at 98°C.

Vortex the samples vigorously and remove the beads using magnetic stand.

Subject 70µL of the sample to electrophoresis approximately 2 cm into a Bolt gel (ThermoFisher).

Fix the gel in 50% ethanol and 10% acetic acid for 1h 0m 0s.

Wash the gel several times in dH20, and stain the proteins with colloidal Coomassie blue.

Then excise the entire sample for trypsin digestion and mass spectrometry.

Wash the gel pieces with100millimolar (mM) ammonium bicarbonate (AmB)/acetonitrile (ACN) and reduce with 10millimolar (mM) dithiothreitol (DTT) at 50°C for 0h 45m 0s.

Alkylate the cysteines using 100millimolar (mM) iodoacetamide in the dark for 0h 45m 0s at Room temperature (RT).

Wash the gel bands in 100millimolar (mM) AmB/ACN prior to adding 1µg trypsin (Promega #V5111) for 0h 45m 0s incubation at 37°C.

Collect the peptide containing supernatants into a separate tube.

Wash the gel pieces with gentle shaking in 50% ACN/1% FA at Room temperature for0h 10m 0s, and collect the supernatant in the previous tubes.

Do the final peptide extraction step with 80% ACN/1% FA, and 100% ACN, and collect all supernatant.

Dry the peptides in a speedvac and reconstitute with 5% ACN/0.1% FA in water before injecting into LC-MS/MS.

Analyse the peptides by LC-MS/MS using a Dionex UltiMate 3000 Rapid Separation nanoLC coupled to an Orbitrap Elite Mass Spectrometer (Thermo Fisher Scientific Inc.).

Load the samples onto the trap column, which is 150 μm x 3 cm in-house packed with 3 µm ReproSil-Pur® beads.

The analytical column is a 75 µm x 10.5 cm PicoChip column packed with 3 µm ReproSil-Pur® beads (New Objective, Inc. Woburn, MA).

Keep the flow rate at 300 nL/min.

Ellute all the fractions from the analytical column at a flow rate of 300 nL/min using an initial gradient elution of 5% B from to 0h 5m 0s, transition to 40% over 1h 40m 0s, 60% for 0h 4m 0s, ramping up to 90% B for 0h 3m 0s, holding 90% B for 0h 3m 0s, followed by re-equilibration of 5% B at 0h 10m 0s with a total run time of 2h 0m 0s.

Record the mass spectra (MS) and tandem mass spectra (MS/MS) in positive-ion and high-sensitivity mode with a resolution of ∼60,000 full-width half-maximum.

Select the 15 most abundant precursor ions in each MS1 scan for fragmentation by collision-induced dissociation (CID) at 35% normalized collision energy in the ion trap.

Dynamically excluded the previously selected ions from re-selection for 0h 1m 0s. Store the collected raw files spectra in. raw format.

Identify the proteins from the MS raw files using the Mascot search engine (Matrix Science, London, UK. version 2.5.1).

Search the MS/MS spectra against the SwissProt mouse database.

Include carbamidomethyl cysteine as a fixed modification and oxidized methionine, deamidated asparagine and aspartic acid, and acetylated N-terminal as variable modifications in all searches.

Allow three missed tryptic cleavages. Apply a 1% false discovery rate cutoff at the peptide level.

Consider only proteins with a minimum of two peptides above the cutoff for further study.

Visualize the identified peptides/protein by Scaffold software (version 5.0, Proteome Software Inc., Portland, OR).

To estimate BAR enrichment, apply 1µL of bead eluent to a methanol activated polyvinylidene difluoride (PVDF) membrane and then allow to dry completely.

Reactivate the membrane then in methanol, rinse with water, and post-fix in 4% PFA for 0h 30m 0s.

Rinse the blots with TBST (refer materials section) and block with buffer containing either BSA (TBST and 5% BSA) or non-fat milk (TBST and 5% non-fat milk) for detection of biotin or αsyn , respectively.

Detect the biotinylated proteins by ABC (VectorLabs) diluted 1:10 in BSA blocking buffer for 1h 0m 0s at Room temperature.

Αsyn can be detected using SYN1 (BD Biosciences) diluted 1:2,000 and PSER129 detected using EP1536Y diluted 1:50,000 both diluted in non-fat milk blocking buffer.

Detect the primary antibodies by incubating blots for 1h 0m 0s in secondary anti-mouse HRP conjugate diluted 1:6,000 or secondary anti-rabbit HRP conjugate (Cell signaling) diluted in milk blocking buffer.

Following secondary antibody, wash the membranes in high stringency wash buffer (Refer materials section) and image using enhanced chemiluminescence (ECL) substrate (Biorad, product # 1705060) and Chemidoc imager (Biorad).