TITLE: APEX2 labelling and screening for biotinylated proteins by proteomics
AUTHORS: Chuyu Chen, Ciarra Smith, Loukia Parisiadou

[DESCRIPTION]
Enzyme-catalyzed proximity labeling (PL) combined with mass spectrometry (MS) has emerged as a revolutionary approach to reveal the subcellular proteomes. APEX (engineered ascorbate peroxidase) catalyzes the oxidation of biotin-phenol to the short-lived biotin-phenoxyl radical in the presence of hydrogen peroxide. This radical reacts with electron-rich amino acids such as tyrosine on neighboring proteins resulting in their biotinylation. APEX2 technology uses a mutant enzyme that increases sensitivity. This protocol describes APEX2 labelling and sample procurement for mass spectrometry from SNc and striatum. 

[STEPS]
SECTION: APEX2 Labelling
1. Construct: AAV5-CAG-DIO-APEX2NES is injected into the ventral midbrain (VM) of DATIRES-Cre mice
SECTION: APEX2 Labelling
2. Confirmation of cellular specificity: Representative slices of SN and Str stained for TH, V5 and merged
SECTION: APEX2 Labelling
3. Brains perfused with aCSF by trascardial perfusion with 10ml ice cold cutting solution
SECTION: APEX2 Labelling
4. Acute slice preparation as 300μm coronal slices
SECTION: APEX2 Labelling
5. Incubation of slices with 0.5mM BP in oxygenated artificial cerebrospinal fluid (aCSF) during the slice recovery period (1hr RT)
SECTION: APEX2 Labelling
6. Rapid biotinylation:  rapid labeling with 1 mM H2O2 in aCSF for 3 minutes
SECTION: APEX2 Labelling
7. Rapid quenching by transferring slices to quenching aCSF (20mM Sodium ascorbate, 10mM NaN3 and 10mM Trolox) for 5 minutes.
SECTION: APEX2 Labelling
8. Slices were transferred to ice-cold quenching aCSF for rapid dissection, tissue were flash frozen and stored in -80C for downstream western blot or proteomics.     Tissue lysis and capture biotinylated proteins for proteomics
SECTION: Tissue lysis and capture of biotinylated proteins for proteomics
9. Frozen tissues or synaptosome pellets were homogenized on ice in a glass dounce homogenizer with 30 strokes (in 750ul lysis buffer with protease and phosphatase inhibitor). One mouse/sample for striatum and 2mice/sample for SNc.
SECTION: Tissue lysis and capture of biotinylated proteins for proteomics
10. Lysates with addition 39ul of 10% SDS were rotated for 15 min at 4°C
SECTION: Tissue lysis and capture of biotinylated proteins for proteomics
11. Lysates were clarified by centrifugation at 21,000 × g for 10 min at 4°C
SECTION: Tissue lysis and capture of biotinylated proteins for proteomics
12. Supernatants were transferred to a new prechilled Eppendorf tube for trichloroacetic acid (TCA) precipitation (for MS)
SECTION: Tissue lysis and capture of biotinylated proteins for proteomics
13. Proteins were precipitated from lysates by the addition of an equal volume of ice-cold 55% TCA.
SECTION: Tissue lysis and capture of biotinylated proteins for proteomics
14. Samples were incubated on ice for 15 min, followed by centrifugation at 21,000 × g for 10 min at 4°C.
SECTION: Tissue lysis and capture of biotinylated proteins for proteomics
15. Protein pellets were resuspended in 1 ml of acetone prechilled to −20 °C and recentrifuged. Pellets were resuspended and recentrifuged another three times in 1 ml of acetone prechilled to −20°C, for a total of four washes
SECTION: Tissue lysis and capture of biotinylated proteins for proteomics
16. Protein pellets were resuspended in Urea Dissolve Buffer (with protease and phosphatase inhibitor) and sonicate for 10 second followed by gentle agitation on an orbital shaker for 1 hr at room temperature.
SECTION: Tissue lysis and capture of biotinylated proteins for proteomics
17. Streptavidin magnetic beads (Thermo Fisher #88817) were resuspended and washed three times in Urea Detergent Wash Buffer for 10 min at 4°C. After washing, streptavidin beads were resuspended in ice-cold Urea Detergent Wash Buffer and 50 μl containing 0.5 mg of beads was added to each sample.
SECTION: Tissue lysis and capture of biotinylated proteins for proteomics
18. Proteins were incubated with streptavidin beads overnight on a rotor at 4°C for 14-18hrs.
SECTION: Tissue lysis and capture of biotinylated proteins for proteomics
19. Beads were washed three times for 5–10 min in 1 ml of Urea Detergent Wash Buffer at room temperature. After the third wash, beads were resuspended in 1 ml of Urea Wash Buffer and transferred to a new tube
SECTION: Tissue lysis and capture of biotinylated proteins for proteomics
20. After three 5–10 min washes in 1ml  Urea Wash Buffer at room temperature, beads were resuspended in 200 μl of Urea Wash Buffer and transferred to a new tube
SECTION: Tissue lysis and capture of biotinylated proteins for proteomics
21. 10 μl aliquot (5%) was transferred to a separate tube for western blotting, and the remaining 190 μl of buffer were removed on a magnetic stand. Beads were flash frozen and stored at −80°C