TITLE: APEX2-based proximity biotinylation of NLRP3 and P4C (SidC) during inflammasome activation
AUTHORS: Tao_Fu, Harper JW, Louis R R Hollingsworth, sharan_swarup

[DESCRIPTION]
APEX2-based proximity labeling for the discovery of proteins proximal to NLRP3. Aspects of this APEX2 protocol can be applied to other target proteins.

[STEPS]
SECTION: Cell line construction and validation (brief overview)
1. The choice of cells, constructs, proximity biotinylation enzyme (e.g., TurboID vs. APEX2), treatments, etc. to employ for proximity biotinylation experiments depends on the experimental design. Here, mouse immortalized bone-marrow derived macrophages iBMDM cells (Gift from Kate Fitzgerald), either wild-type or Nlrp3-knockout, were reconstituted with Nlrp3-APEX2-FLAG-IRES-GFP (Addgene #218633) or FLAG-APEX2-P4C-IRES-mNeonGreen (Addgene #218646). Adherent cells are important for the washing steps of this protocol, but it can be adapted for suspension cells with the addition of spin steps during quenching. 



SECTION: APEX2 proximity biotinylation
8.1. Add 10 μL biotin phenol (BP, 500 mM) to cells 45 mins prior to collecting them. For example, for 4 h LPS priming only, add BP to plates 3 h 15 min after LPS. For a 30 min nigericin treatment, add BP to plates 3 h 45 min after LPS treatment, nigericin at 4 h LPS, and collect cells 30 min later.

 

SECTION: Cell line construction and validation (brief overview)
1.1. These cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM, high glucose and pyruvate) supplemented with 10% fetal bovine serum, GlutaMAX‱, and Penicillin-Streptomycin. They were maintained in a 5% CO2 incubator at 37 oC. iBMDMs were split 1:10 every other day, but growth rates vary among different cell types and passages.

SECTION: Cell line construction and validation (brief overview)
1.2. The appropriate iBMDMs were spinfected with Nlrp3-APEX2-FLAG-IRES-GFP (Addgene #218633) or FLAG-APEX2-P4C-IRES-mNeonGreen (Addgene #218646). 3 - 5 d after spinfection, cells were bulk sorted for low GFP+/mNeonGreen+ expressors with a Sony SH800 Cell Sorter operating in purity mode.

 


SECTION: Cell line construction and validation (brief overview)
2. Check protein expression levels by comparing LPS-primed WT iBMDMs to LPS-primed Nlrp3-APEX2-FLAG iBMDMs using Western blot.
SECTION: Cell line construction and validation (brief overview)
3. Check protein localization by IF using an anti-FLAG antibody.

 

SECTION: Cell line construction and validation (brief overview)
4. Validate functional reconstitution of Nlrp3-APEX2 in Nlrp3-KO iBMDMs with LDH release.

 

SECTION: APEX2 proximity biotinylation
6. Grow cells to just fully confluent in 10-cm dishes. For certain iBMDMs, for example, a 1:10 split produces a fully confluent dish 2 d later. Cells should be seeded in 10 mL complete DMEM.
SECTION: APEX2 proximity biotinylation
7. For the cell harvesting day, freshly prepare: 100X Trolox in DMSO (500 mM, requires sonication), 100X Sodium ascorbate in PBS (1 M), 100X sodium azide in PBS (1 M). Each of these solutions is 1:100X, so prepare enough for the amount of quenching buffer used (35 mL buffer per replicate).
SECTION: APEX2 proximity biotinylation
9. Just before harvesting the cells, prepare the following: 


SECTION: APEX2 proximity biotinylation
5. Prepare the following ahead of time: Biotin-phenol/biotin tyramide (500 mM in DMSO, aliquot, store at -80 °C)
SECTION: APEX2 proximity biotinylation
8. Stagger treatments so that only 3-4 plates (for example, 1 group of replicates) are harvested at a time. For inflammasome treatments, this could mean staggering LPS treatments for different conditions by 1 h. Regardless, replace the medium with 10 mL fresh DMEM/etc.

 

SECTION: APEX2 proximity biotinylation
9.1. 100 mM H2O2 in DPBS (10 uL of 30% H2O2 into 990 μL DPBS). Prepare individual pipettes with 100 uL of H2O2 for each plate being harvested at a time. 
SECTION: APEX2 proximity biotinylation
9.2. 3 falcon tubes per plate, with 10 mL ice-cold quenching buffer in each. 


 

SECTION: APEX2 proximity biotinylation
9.3. Trays of ice for harvesting cells.
SECTION: APEX2 proximity biotinylation
10. Bring cells to the bench and harvest them (see video and text below).


 

 

SECTION: Biotin-streptavidin immunoprecipitation and on-bead digestion
11. Prepare lysis buffer: 50 mM Tris HCl pH 7.5, 150 mM NaCl, 1.0% (v/v) SDS, 1.0 % (v/v) NP-40, 1X cOmplete‱ Protease Inhibitor Cocktail
 

SECTION: APEX2 proximity biotinylation
10.5. Repeat with additional replicates at staggered timepoints to prevent an overwhelming amount of simultaneous bench work.
SECTION: APEX2 proximity biotinylation
10.2. Pour off the solutions and quickly add ice-cold quencher solution to each plate, and place them on ice. 
SECTION: APEX2 proximity biotinylation
10.3. Aspirate the quencher and wash the plates two additional times. Scrape the cells in the final 10 mL quencher solution, transfer to a falcon tube, and scrape the plates with an additional 5 mL quencher solution to harvest residual cells. 
SECTION: APEX2 proximity biotinylation
10.4. Centrifuge cells (1000 xg, 5 min), aspirate the quencher solution, and flash-freeze pellets in liquid nitrogen. Store at -80 oC until ready for immunoprecipitation.
SECTION: Biotin-streptavidin immunoprecipitation and on-bead digestion
12. Rapidly thaw cell pellets by submerging tubes in warm water for ~30 s, then place them on ice.
SECTION: Biotin-streptavidin immunoprecipitation and on-bead digestion
13. Gently vortex the stock of magnetic streptavidin beads for ~30 s to thoroughly resuspend them. Aliquot 50 μL beads (slurry) per replicate, and ~10% excess for pipetting loss, into a protein LoBind microcentrifuge tube using a wide bore P1000 pipette tip. Wash 3x with lysis buffer, and then rotate end-over-end while preparing other reagents.
SECTION: APEX2 proximity biotinylation
10.1. Quickly pipette H2O2 into each of the plates, swirl, and incubate at RT for 1 min. 
SECTION: Biotin-streptavidin immunoprecipitation and on-bead digestion
14. Add 1 mL of lysis buffer to each cell pellet and immediately transfer them to microcentrifuge tubes at RT.

 

SECTION: Biotin-streptavidin immunoprecipitation and on-bead digestion
15. Sonicate each lysate for ~10 seconds with a small probe sonicator. 

SECTION: Biotin-streptavidin immunoprecipitation and on-bead digestion
15.1. Repeat this step once.
SECTION: Biotin-streptavidin immunoprecipitation and on-bead digestion
16. Add 0.25 μL benzonase to each tube. Invert tubes at RT for 30 min to complete lysis and DNA digestion. The solutions should be much less viscous following sonication and benzonase treatment.
SECTION: Biotin-streptavidin immunoprecipitation and on-bead digestion
17. Spin tubes at 15,000 xg for 10 min. There shouldn't be a large pellet.
SECTION: Biotin-streptavidin immunoprecipitation and on-bead digestion
18. Quantify each of the supernatants with the DC protein assay kit II. Use reagent A'.
SECTION: Biotin-streptavidin immunoprecipitation and on-bead digestion
18.1. For a fully confluent 10 cm plate lysed in 1 mL, dilute the clarified lysates 1:5 in lysis buffer to be in the linear range of the assay.
SECTION: Biotin-streptavidin immunoprecipitation and on-bead digestion
18.2. Run a BSA standard curve alongside the APEX2 lysates by diluting 200 mg/mL analytical standard BSA solution to 4 mg/mL in lysis buffer. Perform several 2-fold serial dilutions to make a 6-point curve (4, 2, 1, 0.5, 0.25, 0.125 mg/mL)
SECTION: Biotin-streptavidin immunoprecipitation and on-bead digestion
19. Normalize protein concentrations to 2 mg/mL. Prepare a total volume of 1075 μL in fresh protein LoBind microcentrifuge tubes. Remove 75 μL of this solution into pre-prepared microcentrifuge tubes containing 25 μL 4XLDS sample buffer ("INPUT" sample).
  

SECTION: Biotin-streptavidin immunoprecipitation and on-bead digestion
20. Add 50 μL of bead slurry to each microcentrifuge tube containing 1 mL clarified lysate at 2 mg/mL. Resuspend or invert the beads constantly to ensure they do not settle to the bottom during aliquotting. Invert the mixtures end-over-end for 2 h at RT.

 

SECTION: Biotin-streptavidin immunoprecipitation and on-bead digestion
21. Wash the beads thoroughly with several solutions, using a magnetic rack to pellet the beads. Resuspend the magnetic beads with a wide bore pipette when changing solutions and place them on an end-over-end rotator while resuspending all of the replicates. Ensure that the beads don't aggregate, and if they do, gently pipette up and down with a wide bore P1000 to disperse them.
SECTION: Biotin-streptavidin immunoprecipitation and on-bead digestion
21.1. Wash 1: 1 mL lysis buffer
SECTION: Biotin-streptavidin immunoprecipitation and on-bead digestion
21.2. Wash 2: 1 mL lysis buffer
SECTION: Biotin-streptavidin immunoprecipitation and on-bead digestion
21.3. Wash 3: 1 mL 1 M KCl
SECTION: Biotin-streptavidin immunoprecipitation and on-bead digestion
21.4. Wash 4: 1 mL 100 mM Sodium carbonate (Na2CO3)
SECTION: Biotin-streptavidin immunoprecipitation and on-bead digestion
21.5. Wash 5: 1 mL 2 M urea in 10 mM Tris-HCl (pH 8.0)
SECTION: Biotin-streptavidin immunoprecipitation and on-bead digestion
21.6. Wash 6: 1 mL PBS. Use this wash to transfer the beads to new protein low-bind tubes.

 

SECTION: Biotin-streptavidin immunoprecipitation and on-bead digestion
21.7. Wash 7: 1 mL MQ/ultrapure water. 

 

SECTION: Biotin-streptavidin immunoprecipitation and on-bead digestion
22. Reduction: pellet the beads and resuspend them in 75 μL EPPS pH 8.5 (200 mM) with 1.5 μL TCEP (0.5 M, ~10 mM final). Vortex, and incubate tubes at RT for 30 min.
SECTION: Biotin-streptavidin immunoprecipitation and on-bead digestion
23. Alkylation: add 3 μL of freshly prepared 0.5 M chloroacetamide (in water) to each tube (final: ~20 mM). Vortex and incubate for 20 min in the dark (30 minutes max, and longer is bad).
SECTION: Biotin-streptavidin immunoprecipitation and on-bead digestion
24. Quench the chloroacetamide with DTT (3.5 μL, 0.5 M), vortex, and pellet on a magnetic rack.
SECTION: Biotin-streptavidin immunoprecipitation and on-bead digestion
25. Wash beads 2x with 1 mL HPLC water to remove chloroacetamide, TCEP, and DTT.
SECTION: Biotin-streptavidin immunoprecipitation and on-bead digestion
26. Resuspend beads in 75 μL EPPS supplemented with 1 μL LysC. Shake (600-800 RPM) for 2-4 h at 37 oC (ideally, with a heated lid to prevent evaporation).
SECTION: Biotin-streptavidin immunoprecipitation and on-bead digestion
27. Add 1 μL trypsin per tube. Vortex briefly and shake (600-800 RPM) for 16-24 h at 37 oC (ideally, with a heated lid to prevent evaporation).
SECTION: Mass spec prep
28. Vortex beads, pulse centrifuge them, and then put them on a magnetic rack. Remove the digested peptide flow through to a new low-bind microcentrifuge tube.
SECTION: Mass spec prep
29. Wash the beads with 30 μL acetonitrile (ACN), and combine the flow through into the new tube.
SECTION: Mass spec prep
30. Pellet the combined flow-through on a magnetic rack and take the flow-through, avoiding additional bead carry-over.
 

SECTION: Mass spec prep
31. Add 5 μL TMT label reagent (12.5 μg/μl in ACN) to each vial. Vortex and then shake (600-800 RPM) for 2 h at RT.
SECTION: Mass spec prep
32. In the last 30 min of the TMT labeling, prepare a stage tip (6 small cookies from Empore SPE C18 disks, or a commercial C18 column) and equilibrate it:

 

SECTION: Mass spec prep
32.1. 200 μL 100% Methanol

SECTION: Mass spec prep
32.3. 200 μL 5% ACN/1% Formic acid (FA)
SECTION: Mass spec prep
32.2. 200 μL 80% ACN

SECTION: Mass spec prep
33. Place 160 μL of 1% FA in a microcentrifuge tube. Pipette 5 μL (~5 % of output) of each labeled sample into tube, vortex, and then proceed immediately to stage tip. Place remaining TMT-labeled peptides at -20 oC until after a ratio check.
SECTION: Mass spec prep
34. After the sample flows through the stage tip, wash it with 200 μL 5% ACN/1% FA.

SECTION: Mass spec prep
34.1. Elute the stage tip into an MS vial with 80 μL of 80% ACN
SECTION: Mass spec prep
35. Dry the sample in a speed vacuum and then resuspend the dried peptides in 5% ACN/5% FA
SECTION: Mass spec prep
36. Perform a ratio check search with MS. Ensure that TMT labeling is >95%, otherwise add more TMT labeling reagent to each tube and repeat the stage tip + ratio check.
SECTION: Mass spec prep
37. Quench each tube with hydroxylamine at a final concentration of 0.5% (wt/v). With the volumes above, add 1.1 μL of 50% (wt/v) hydroxylamine solution. Vortex then incubate at RT for 15 min.
SECTION: Mass spec prep
38. Pool equal amounts of peptides from each channel depending on the ratio check search. Note potential outliers from the ratio check search (e.g., one sample that was not represented, or was very low input).

 

SECTION: Mass spec prep
40. Fractionate the sample with the High pH Reversed-Phase Peptide Fractionation Kit (Thermo 84868). Follow most of the manufacturer's instructions, but elute with the following 12 fractions:
SECTION: Mass spec prep
39. Dry samples in the speed vacuum. This will likely take several hours (> 4 h).
SECTION: Mass spec prep
40.1.  
 
 
SECTION: Mass spec prep
41. Combine fractions 1 and 7; 2 and 8; 3 and 9; 4 and 10; 5 and 11; 6 and 12. This concatenates the sample space to 6 fractions.
SECTION: Mass spec prep
42. Dry these fractions on a speed vacuum. They will dry at different rates, and likely take between 4 and 8 h.
SECTION: Mass spec prep
43. Resuspend samples in 200 μL of 3% ACN/1% FA. Check that pH is ~2, otherwise, further acidify with small amounts of 100% FA.
SECTION: Mass spec prep
44.1. 200 μL 100% Methanol
SECTION: Mass spec prep
44. Prepare 6 stage tips (12 small cookies from Empore SPE C18 disks, or a commercial C18 column) and equilibrate them:
SECTION: Mass spec prep
44.5. 200 μL 5% ACN/1% Formic acid (FA)
SECTION: Mass spec prep
44.4. Load sample. Collect this flow-through and save it at -20.
SECTION: Mass spec prep
44.3. 200 μL 5% ACN/1% Formic acid (FA)
SECTION: Mass spec prep
44.2. 200 μL 80% ACN
SECTION: Mass spec prep
45. Dry these stage-tipped fractions on a speed vacuum. They will dry at different rates, and likely take between 2 and 4 h.
SECTION: Mass spec prep
44.6. Elute stage tips into an MS vials with 80 μL of 80% ACN
SECTION: Mass spec prep
46. Resuspend the dried peptides in 8 μL of 5% ACN/5% FA, and proceed to MS.