APEX2 biotinylation in brain slices

APEX2 Mouse Brain Acute Slice Proteomics

Part 1: Tissue Labeling

Benjamin D. Hobson

Developed in the laboratories of Dr. Peter Sims and Dr. David Sulzer, Columbia University

This protocol (Part 1) describes steps in tissue processing in order to isolate biotinylated proteins from APEX2-labeled, frozen mouse brain tissue. The starting point is frozen brain tissue stored at -80C (see Part 2 for details on tissue processing after labeling). The major steps are:

1. Preparation of acute brain slices from mice expressing the APEX2 construct of choice
2. Slice recovery and incubation with biotin phenol
3. Acute labeling via the addition of hydrogen peroxide
4. Quenching of labeling, further tissue dissection (if required) and tissue freezing

The frozen tissue can then be stored at -80C prior to proceeding onto the protocol for tissue processing (see Part 2).

Please cite the original paper Hobson et al. (2022) in your methods section:

Hobson, B. D., Choi, S. J., Mosharov, E. V., Soni, R. K., Sulzer, D., & Sims, P. (2022). Subcellular proteomics of dopamine neurons in the mouse brain. ELife, 11, e70921. https://doi.org/10.7554/eLife.70921

Buffers and solutions:

NMDG cutting solution

92 mM N-Methyl-D-glucamine/NMDG

2.5 mM KCl

30 mM NaHCO3

20 mM N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES)

1.25 mM NaH2PO4

2 mM thiourea

5 mM sodium ascorbate

3 mM sodium pyruvate

10 mM MgSO4

0.5 mM CaCl2

25 mM d-glucose

Artificial cerebrospinal fluid (aCSF)

125.2 mM NaCl

2.5 mM KCl

26 mM NaHCO3

1.3 mM MgCl2·6H2O

2.4 mM CaCl2

0.3 mM NaH2PO4

0.3 mM KH2PO4

10 mM d-glucose

Biotin tyramide (Biotin Phenol/BP) – Iris Biotech GmbH, cat #LS3500

Store aliquots at 500mM (1000x) in DMSO at -80C.

Hydrogen Peroxide (H2O2) – MilliporeSigma, cat #H1009

30% w/w Hydrogen peroxide = 9.8M ~ 10,000X

Store at 4C, tightly sealed and wrapped in foil. Carefully and quickly remove solution at time of experiment using a fresh serological pipette.

Dilute once at 1:10 in H2O, then use 1:1000 directly into the aCSF.

Trolox – (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid

MilliporeSigma, cat #238813-5G

Sodium azide (NaN3)

Sodium ascorbate

Tissue Labeling Protocol:

Basic knowledge of acute brain slice preparation methods and materials is assumed.

For adult mice, we employ a modified cutting solution (with NMDG), please see the following protocol:

https://www.jove.com/v/53825/preparation-acute-brain-slices-using-an-optimized-n-ethyl-d

Acute brain slice preparation

1. Anesthetize mice with euthasol and transcardially perfuse with 10–15 ml of ice-cold cutting solution at pH 7.3–7.4 and saturated with 95% O2/5% CO2.
2. Rapidly extract brains and place into ice-cold cutting solution saturated with 95% O2/5% CO2. Coronal or sagittal slices are then glued directly to the vibratome stage (Leica VT1000S).
3. Cut 300-µm-thick slices containing the brain regions of interest in ice-cold cutting solution continuously saturated with 95% O2/5% CO2.

APEX2 biotinylation in brain slices

1. After preparation in cold cutting solution, transfer slices to jars containing 70 ml of aCSF supplemented with 0.5 mM BP (note: 1 µM tetrodotoxin can be optionally included to further preserve neuronal viability in cases where physiological manipulation is not desired)
2. Continuously saturated aCSF with 95% O2/5% CO2. Allow slices to recover for 60 min at room temperature.
3. After recovery, initiate APEX2 labeling by the addition of 1 mM H2O2 to aCSF at room temperature (1:1000 volume of 0.98 M H2O2, see above).
4. The length of H2O2 labeling period can be varied depending on the experimental goal. We found 3 min to be sufficient for downstream proteomics applications without causing notable structural changes to axons as assessed by immunostaining.
5. To quench labeling, rapidly transfer slices to a separate jar containing 50 ml of quenching aCSF (aCSF supplemented with 10 mM Trolox, 20 mM sodium ascorbate, and 10 mM NaN3).
6. After 5 min rest at room temperature, transfer slices to either fixative solution of your choice (e.g., 4% PFA) for downstream immunostaining, or to ice-cold quenching aCSF for rapid dissection and downstream western blotting/proteomics.
7. For proteomics, transfer dissected tissue into 1.5 mL Eppendorf tubes, cap tightly, and immediately flash freeze in liquid nitrogen. Store at -80C until tissue processing.

APEX2 Mouse Brain Acute Slice Proteomics

Part 2: Tissue Processing

Benjamin D. Hobson

Developed in the laboratories of Dr. Peter Sims and Dr. David Sulzer, Columbia University

This protocol (Part 2) describes steps in tissue processing in order to isolate biotinylated proteins from APEX2-labeled, frozen mouse brain tissue. The starting point is frozen brain tissue stored at -80C (see Part 1 for details on APEX2 labeling procedures in acute slices from mouse brain). The major steps are:

    5. Tissue lysis (with anti-oxidants and sodium azide to prevent further APEX2 activity)

    6. Protein precipitation in order to remove soluble biotin and lipids

    7. Streptavidin bead purification of biotinylated proteins

    8. Alkylation, reduction, and freezing of proteins on streptavidin beads 

The protocol requires two days to complete. The frozen beads can then be stored at -80C prior to submitting for on-bead tryptic digest and mass spectrometry at a proteomics core facility.

This protocol was adapted from Kalocsay (2019) and described in Hobson et al. (2022)

Please cite both papers in your methods section:

Hobson, B. D., Choi, S. J., Mosharov, E. V., Soni, R. K., Sulzer, D., & Sims, P. (2022). Subcellular proteomics of dopamine neurons in the mouse brain. ELife, 11, e70921. https://doi.org/10.7554/eLife.70921

Kalocsay, M. (2019). APEX Peroxidase-Catalyzed Proximity Labeling and Multiplexed Quantitative Proteomics. Methods in Molecular Biology (Clifton, N.J.), 2008, 41–55. https://doi.org/10.1007/978-1-4939-9537-0_4

Buffers:

Tissue Lysis Buffer

50mM Tris pH 8.0, 150mM NaCl, 10mM EDTA, 1% Triton X-100, 0.5% SDS, 10 mM sodium ascorbate, 5mM Trolox, 10mM sodium azide

100% Trichloroacetic acid (TCA) – Sigma, cat #T6399-500G

Add 227 mL of MQ H₂O to an unopened 500g bottle of TCA. Once dissolved, store in dark at 4C. Make dilutions freshly for precipitation.

Urea Stock Buffer (8M Urea, 100 mM sodium phosphate pH 8) -- stock prepared fresh each day

For 50mL, add 24.02g of Urea, 668mg of dibasic sodium phosphate, and 36 mg of monobasic sodium phosphate. Dissolve in ~40 mL MQ H₂O, heat at 37C for ~30min. Check pH and adjust volume to 50 mL with MQ H₂O.

Urea Dissolve Buffer

8M Urea, 1% SDS, 100 mM sodium phosphate pH 8, 100mM NH₄HCO₃

For 15 mL, dissolve 118.6 mg of ammonium bicarbonate and 150 mg of SDS in Urea Stock Buffer

Urea Detergent Wash Buffer

4M Urea, 0.5% SDS, 100mM sodium phosphate pH 8

For 50 mL, dissolve 250 mg SDS in 25 mL of MQ H₂O and add to 25 mL of Urea Stock.

Urea Wash Buffer

4M Urea, 50 mM sodium phosphate pH 8

Dilute Urea Stock buffer 1:1 with MQ H₂O.

Elution Buffer (3x LDS Sample Buffer + 2mM biotin + 20mM DTT)

Make 1 mL of 20 mM biotin—resuspend 5 mg biotin in 1 mL of MQ H₂O.

Final = 750 uL of 4x LDS SB + 100 uL of 20 mM biotin + 20 uL of 1M DTT + 130 uL MQ H₂O

Iodoacetamide Stock (20x) – ThermoFisher, cat. #90034

400 mM Iodoacetamide in 50 mM NH₄HCO₃

For 1 mL, dissolve 7.9 mg of ammonium bicarbonate and 74 mg of iodoacetamide in MQ H₂O

Must be made fresh day of experiment

1M DTT Stock (20x)

To make 10 mL of 1M DTT, dissolve 154.3mg of DTT in 1 mL MQ H₂O. Store aliquots frozen at -20C or make fresh.

500 mM TCEP Stock (50x) – Sigma, cat. #646547

Open a fresh vial immediately prior to adding to samples.

Tissue Processing Protocol:

Day 1: Tissue Lysis, Protein Purification, IP Setup

Lyse tissues directly from -80C, only allowing a brief moment in the dounce homogenizer in 4C lysis buffer.

Striatal Tissue

1. Lyse tissue in 750 uL of tissue lysis buffer in dounce homogenizer (30 strokes in dounce A and B, take care not to create foaming). Rotate lysates for 15 minutes at 4C to ensure complete lysis of axons.
2. Spin 1,000xg at 4C for 10 minutes to clear nuclei and cellular debris.
3. Transfer supernatant to a fresh tube and add 1/20 the volume of 10% SDS (0.5% final concentration; should be 30-40 uL. Vortex for a few seconds and rotate at 4C for 15 minutes.
4. Reserve 5% volume of the lysate (30-40uL) as input and freeze at -80C.
5. Proceed to TCA precipitation.

Midbrain Tissue

1. Lyse tissue in 750 uL of tissue lysis buffer in dounce homogenizer (30 strokes in dounce A and B, take care not to create foaming). Add 1/20 the volume of 10% SD (0.5% final concentration). Vortex for a few seconds and rotate at 4C for 15 minutes to completely lyse cells (including nuclei).
2. Proceed to TCA precipitation.

TCA Precipitation

1. Prepare 55% TCA from 100% TCA stock (roughly 1 mL per sample). For 6 mL, 2.75 mL of 100% TCA to 3.25 mL of MQ H₂O. Chill on ice.
2. Add 1:1 ice-cold TCA stock solution 1:1 to lysate and incubate on ice for 15 min.
3. Pellet precipitated proteins at max speed (21,130xg) at 4C for 10 min.
4. Wash the pellet with -20C-cold acetone, vortex, and spin again 21,130xg | 4C | 10min.
5. Conduct 3 more washes with -20C-cold acetone. After the last wash, use a P20 to remove residual acetone.
6. Resuspend pellets in 686 uL of Urea Dissolve Buffer. Tubes can be incubated in the sonication water bath or on a vertical rocker. Check pH is >7.5 (no residual TCA).
7. Spin dissolved proteins at 21,130xg at room temperature for 10 min, transfer supernatant to clean tube. 

Cysteine Alkylation and Streptavidin Pull-Down

1. Note the volume of protein suspension at this point and adjust volumes accordingly
2. Add 1/49 volume of TCEP 50x stock (~14 uL) to the protein suspension.
3. Add 1/19 volume of Iodoacetamide 20x stock (~36.8 uL) to the protein suspension.
4. Vortex briefly and incubate in the dark for 25 minutes at room temperature
5. To quench alkylation, add 1/19 volume of 20x DTT stock (~38.8 uL).
6. Add 0.87x volume of MQ H₂O of the original protein suspension volume (from step 1). This is to dilute samples to final IP concentrations of Urea Dissolve reagents (0.5x = 4M Urea, 0.5% SDS, 50 mM sodium phosphate)
7. Wash the appropriate volume of Pierce Streptavidin beads (see below) twice with >1mL of Urea Detergent Wash Buffer.
8. After the second wash, resuspend the streptavidin beads in ~50 uL of Urea Detergent Wash Buffer.
9. Add streptavidin beads to protein suspension and incubate on rotor at 4C overnight. 

Volumes breakdown:

Urea Dissolve Buffer (686 uL)

50x TCEP (14 uL)

20x iodoacetamide (36.84 uL)

20x DTT (38.78 uL)

597 uL H2O

= 1372 uL (4M Urea, 0.5% SDS, 50 mM sodium phosphate) + 50 uL of Streptavidin beads in Urea Detergent Wash Buffer

Pierce Streptavidin Beads (10 mg/ml)

Binding capacity is ~55 ug of biotinylated rabbit IgG per mg of beads

This is approximately 100 uL for ~55 ug protein, so ~1.8 uL beads per ug protein

Typically, ~50 uL beads for VM and ~75 uL beads for Str

Day 2: Cleanup of Pull-Down and Freezing of Beads for On-Bead Digestion

Note the tube changes and lack of SDS in the wash series, this is critical, as residual SDS will cause serious problems in downstream proteomics applications.

1. Remove beads and protein suspension from 4C rotor and collect beads on magnet. The protein suspension can be discarded; alternatively, save to check for residual biotinylated proteins by western blot.
2. Resuspend beads in >1mL of Urea Detergent Wash Buffer and immediately transfer to a fresh tube. Wash beads at room temperature for 5 minutes, a total of three times.
3. Resuspend beads in >1 mL of Urea Wash Buffer (no detergent!) and immediately transfer to a fresh tube. Wash beads at room temperature for 5 minutes, a total of three times.
4. After the third was in Urea Wash Buffer, resuspend beads in 100 uL (or 200 uL) of Urea Wash Buffer and immediately transfer to a fresh tube.
5. Aliquot 10 uL (or 20 uL) of the resuspended beads to a separate tube (10% of the IP material) for use in western blot. These beads can be frozen or eluted by boiling in 3x LDS sample buffer supplemented with 2mM biotin and 20mM DTT at 95C for 5 min.
6. For the remaining 90% of the beads, remove residual Urea Wash Buffer. Immediately flash freeze on dry ice (and store at -80C) or directly proceed to on-bead digest.

APEX Protein IP Buffer Matrix: Sufficient for 10-15 Samples*

APEX Protein IP Buffer Matrix: Sufficient for 10-15 Samples*

* Typical volume of 750 uL lysis buffer per sample
** 1/20 vol (~35 uL) of 10% SDS is added to each sample after homogenization and nuclear clearing spin (1,000xg 10min)