TITLE: Biotinylation by antibody recognition
AUTHORS: Bryan_Killinger

[DESCRIPTION]
This protocol details the biotinylation by antibody recognition.

[STEPS]
SECTION: Biotinylation by antibody recognition
1. 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 60 min each in TBST. 

SECTION: Biotinylation by antibody recognition
2. Place the sections in 0.3% hydrogen peroxide and 0.1% sodium azide diluted in blocking buffer for 60 min at Room temperature to quench endogenous peroxidases.

SECTION: Biotinylation by antibody recognition
3. Rinse the sections briefly in TBST and incubate in anti-PSER129 antibody EP1536Y diluted 1:50,000 in blocking buffer 60 min at 4 °Cwith gentle agitation. 

SECTION: Biotinylation by antibody recognition
4. The following day, wash the sections 3 times in TBST, then incubate with biotinylated anti-rabbit antibody diluted 1:200 in blocking buffer for 60 min at Room temperature

SECTION: Biotinylation by antibody recognition
5. Wash the sections 3 times in TBST, incubate with ABC reagent for 60 min, and wash off with borate buffer.

SECTION: Biotinylation by antibody recognition
6. Incubate the sections with borate buffer containing biotinyl tyramide as described above. 
SECTION: Biotinylation by antibody recognition
7. Wash the sections 60 min with TBST, gather in a 1.5mL Eppendorf tube, 3000 x g, 15 min to pellet floating sections, and discard the supernatant.

SECTION: Biotinylation by antibody recognition
8. Briefly sonicate each sample in 1 mL of crosslink reversal buffer (refer materials section) and heat for 30 min at 98 °C followed by 60 min at 90 °C. 

SECTION: Biotinylation by antibody recognition
9. Centrifuge the sample  20000 x g, 20 min of the samples and then dilute the supernatant 1:10 in modified TBST (refer materials section).

SECTION: Biotinylation by antibody recognition
10. Incubate each sample with 40 mg of streptavidin magnetic beads (Thermofisher Scientific) for 120 min at Room temperature with constant mixing. 

SECTION: Biotinylation by antibody recognition
11. Collect the beads using a magnetic stand (Thermofisher Scientific), wash the beads 3 times in modified TBST, and then 120 min in 10 mL of stringent wash buffer (refer materials section). 

SECTION: Biotinylation by antibody recognition
12. The following day, collect the  beads using magnetic stand and resuspend in 100 µL 1 X Bolt LDS sample buffer with reducing agent (Thermofisher) then heat for 10 min at 98 °C.

SECTION: Biotinylation by antibody recognition
13. Vortex the samples vigorously and remove the beads using magnetic stand. 

SECTION: Biotinylation by antibody recognition
14. Subject 70 µL of the sample to electrophoresis approximately 2 cm into a Bolt gel (ThermoFisher). 

SECTION: Biotinylation by antibody recognition
15. Fix the gel in 50% ethanol and 10% acetic acid for 60 min. 

SECTION: Biotinylation by antibody recognition
16. Wash the gel several times in dH20, and stain the proteins with colloidal Coomassie blue. 
SECTION: Biotinylation by antibody recognition
17. Then excise the entire sample for trypsin digestion and mass spectrometry. 
SECTION: Biotinylation by antibody recognition
18. Wash the gel pieces with100 millimolar (mM) ammonium bicarbonate (AmB)/acetonitrile (ACN) and reduce with 10 millimolar (mM) dithiothreitol (DTT) at 50 °C for 45 min.

SECTION: Biotinylation by antibody recognition
19. Alkylate the cysteines using 100 millimolar (mM) iodoacetamide in the dark for 45 min at Room temperature (RT).

SECTION: Biotinylation by antibody recognition
20. Wash the gel bands in 100 millimolar (mM) AmB/ACN prior to adding 1 µg trypsin (Promega #V5111) for 45 min incubation at 37 °C.

SECTION: Biotinylation by antibody recognition
21. Collect the peptide containing supernatants into a separate tube. 
SECTION: Biotinylation by antibody recognition
22. Wash the gel pieces with gentle shaking in 50% ACN/1% FA at Room temperature for10 min, and collect the supernatant in the previous tubes.

SECTION: Biotinylation by antibody recognition
23. Do the final peptide extraction step with 80% ACN/1% FA, and 100% ACN, and collect all supernatant.
SECTION: Biotinylation by antibody recognition
24. Dry the peptides in a speedvac and reconstitute with 5% ACN/0.1% FA in water before injecting into LC-MS/MS.
SECTION: Biotinylation by antibody recognition
25. 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.).
SECTION: Biotinylation by antibody recognition
26. Load the samples onto the trap column, which is 150 μm x 3 cm in-house packed with 3 µm ReproSil-Pur® beads.
SECTION: Biotinylation by antibody recognition
27. 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).
SECTION: Biotinylation by antibody recognition
28. Keep the flow rate at 300 nL/min.
SECTION: Biotinylation by antibody recognition
29. 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 5 min, transition to 40% over 100 min, 60% for 4 min, ramping up to 90% B for 3 min, holding 90% B for 3 min, followed by re-equilibration of 5% B at 10 min with a total run time of 120 min.

SECTION: Biotinylation by antibody recognition
30. 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. 
SECTION: Biotinylation by antibody recognition
31. 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. 
SECTION: Biotinylation by antibody recognition
32. Dynamically excluded the previously selected ions from re-selection for 1 min. Store the collected raw files spectra in. raw format.

SECTION: Biotinylation by antibody recognition
33. Identify the proteins from the MS raw files using the Mascot search engine (Matrix Science, London, UK. version 2.5.1).
SECTION: Biotinylation by antibody recognition
34. Search the MS/MS spectra against the SwissProt mouse database.
SECTION: Biotinylation by antibody recognition
35. 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.
SECTION: Biotinylation by antibody recognition
36. Allow three missed tryptic cleavages. Apply a 1% false discovery rate cutoff at the peptide level. 
SECTION: Biotinylation by antibody recognition
37. Consider only proteins with a minimum of two peptides above the cutoff for further study. 
SECTION: Biotinylation by antibody recognition
38. Visualize the identified peptides/protein by Scaffold software (version 5.0, Proteome Software Inc., Portland, OR).

SECTION: Biotinylation by antibody recognition
39. To estimate BAR enrichment, apply 1 µL of bead eluent to a methanol activated polyvinylidene difluoride (PVDF) membrane and then allow to dry completely.

SECTION: Biotinylation by antibody recognition
40.  Reactivate the membrane then in methanol, rinse with water, and post-fix in 4% PFA for 30 min.

SECTION: Biotinylation by antibody recognition
41. 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.
SECTION: Biotinylation by antibody recognition
42. Detect the biotinylated proteins by ABC (VectorLabs) diluted 1:10 in BSA blocking buffer for 60 min at Room temperature. 

SECTION: Biotinylation by antibody recognition
43. 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.
SECTION: Biotinylation by antibody recognition
44. Detect the primary antibodies by incubating blots for 60 min in secondary anti-mouse HRP conjugate diluted 1:6,000 or secondary anti-rabbit HRP conjugate (Cell signaling) diluted in milk blocking buffer. 

SECTION: Biotinylation by antibody recognition
45. 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).