Optimized Incorporation of Alkynyl Fatty Acid Analogs for the Detection of Fatty Acylated Proteins using Click Chemistry

1. Cell culture

1. To supplement DMEM (Dulbecco's Modified Eagle Medium) for cell culture, add 10% Fetal Bovine Serum (FBS), 1x Penicillin-Streptomycin, 2 mM L-glutamine, and 100 mM sodium pyruvate (1% vol/vol).
2. Plate approximately 5 x 10⁵ HEK293T cells/well of a 6-well tissue culture dish and grow for 18 h in a 37 °C humidified incubator with 5% CO₂ to reach 75%-80% confluency.
3. Fatty acid serum deprivation
   1. To prepare labeling media, prepare DMEM as above (step 1.1) without 10% FBS. Replace FBS with 5% dextran-charcoal coated FBS (DCC-FBS). Pre-warm to 37 °C before use.
   2. Gently wash the cells with 1x phosphate buffered saline (PBS) at room temperature and replace with labeling media.
      NOTE: HEK293T cells detach from tissue culture plates easily. Take care when washing cells and replacing media. Minimize agitation during transfer to and from the incubator as much as possible.
   3. Return the cells to the 37 °C incubator with 5% CO₂ and incubate approximately 45 min (minimum 15 min is effective), up to 60 min, prior to proceeding with metabolic labeling with fatty acids.

2. Preparation and saponification of fatty acid analogs

1. Prepare the stock solutions of alkynyl fatty acids ahead of time by solubilizing in DMSO to achieve the following concentrations and store at -20 °C. Thaw at room temperature as needed. Store the preparations for their best performance under N₂ or Ar.
   _{Alkynyl-myristate (13-tetradecynoic acid, 13-TDYA): 25 mM
   Alkynyl-palmitate (15-hexadecynoic acid; 15-HDYA): 100 mM
   ​Alkynyl-stearate (17-octadecynoic acid; 17-ODYA): 100 mM}
2. To prepare 20% fatty acid free BSA (FAFBSA), weigh 2 g of FAFBSA in a 50 mL disposable tube.
   1. Bring up to 10 mL with pre-warmed (37 °C) DMEM.
   2. Mix by end-over-end rotation or by vortexing, and place in a 37 °C water bath to dissolve FAFBSA completely.
   3. Use 0.2 µm filter to filter sterilize the medium.
   4. Aliquot in approximately 1 mL volumes and store at -20 °C. Thaw as needed and warm to 37 °C in a water bath prior to use.
3. To enhance the solubility of the fatty acid and fatty acid analogs, saponify by incubating with 20% molar excess of potassium hydroxide (KOH) in 3 mL glass conical reaction vials.
   NOTE: These vials allow the salt to remain soluble while ensuring the FAFBSA does not congeal from the high heat. The use of glass also prevents fatty acids from sticking to the plastic.
   1. Pipette out at least 2 µL of alkynyl fatty acid analog directly to the bottom of a 3 mL conical reaction vial. Prepare 2 µL of lipid per well of a 6-well plate used (see Table 1).
      ​NOTE: Due to the hydrophobicity of fatty acids, it is best to coat the tip of the pipette by drawing up the desired volume several times before dispensing it into the reaction vial.
   2. Dilute 1 M KOH to concentrations equal to 20% molar excess of the alkynyl fatty acid label (30 mM for 13-TDYA and 120 mM for 15-HDYA and 17-ODYA).
   3. Pipette out an equal amount of diluted KOH (1 µL : 1 µL fatty acid : KOH) close to the bottom of the reaction vial on the edge of the glass, such that the dispensed volume of the KOH mixes with the fatty acid.
   4. Close the lid of the vial and tap gently to mix the solutions.
      NOTE: The mixture may solidify quickly, especially with increasing lengths of the hydrocarbon chain of the fatty acid. Be careful not to pipet the solid (i.e., do not mix by pipetting).
   5. Heat the reaction vial at 65 °C for approximately 5 min, or as soon as the fatty acid is incorporated (solution becomes clear).
      NOTE: Fatty acids with a higher number of carbons and decreased solubility, such as stearate (17-ODYA), may require longer incubation times to be fully incorporated intothe KOH at 65 °C. Raise the temperature to 70 °C if needed. A water bath is best. Take care that the liquid does not evaporate too much.
   6. Once the fatty acids have gone into solution and no visible solids remain, pipette prewarmed 20% FAFBSA such that the volume ratio of fatty acids: KOH: FAFBSA is 1: 1: 50 to achieve a final concentration of 20x BSA-bound alkynyl fatty acids.
   7. Mix by pipetting up and down. The solution typically appears clear with no visible solids.
      NOTE: Typically, any small solids will go into solution after incubation at 37 °C.
   8. Incubate the fatty acid and FAFBSA for 15 min at 37 °C.
      NOTE: The saponified label at this point is stable beyond 15 min.
      
       

Table 1: Saponified fatty acid labeling ratios. Experimental volumes of fatty acid, KOH, and FAFBSA for the saponification of fatty acid label according to the volume of media used.

4. As a control, repeat step 2.3. with fatty acids without an alkyne label.
5. Label the cells with 1/20 volume of the 20x fatty acid-BSA conjugate directly onto the starvation media (typically, 100 µL in 2 mL media/cells) to achieve a final concentration of 1% BSA and 25 µM for alkynyl-myristate, or 100 µM for alkynyl-palmitate and alkynyl-stearate.
   NOTE: To minimize the amount of physical disturbance to the attached cells, form a droplet at the tip of the pipette close to the surface of the media instead of pipetting directly into the media. If using acylation inhibitors, add at least 15 min prior to the labeled fatty acids. Times may vary depending on the cells or inhibitor used. It has been recommended to use the saponification for this step as well²⁸.
   1. For comparison, add non-saponified lipids by pipetting 2 µL (or equivalent to volume saponified) of unlabeled fatty acid directly into the starvation media.
   2. Place cells back into the incubator and incubate for 3-6 h.
      NOTE: Optimal labeling timing may need to be determined for each cell type or experimental condition. Longer incubation times can potentially lead to the breakdown of the fatty acids by β-oxidation and/or incorporation into other lipid groups, such as phospholipids²⁸.
6. Gently wash the cells with 1x PBS at room temperature.
7. Harvest and lyse the cells with 500 µL ethylenediaminetetraacetic acid (EDTA)-free modified radioimmunoprecipitation assay (RIPA) buffer (0.1% SDS, 50 mM N-2-hydroxyethylpiperazine-N-ethanesulfonic acid (HEPES) pH 7.4, 150 mM NaCl, 1% non-denaturing detergent, 0.5% sodium deoxycholate, 2 mM MgCl₂ with freshly added 1 mM phenylmethylsulphonyl fluoride (PMSF), and 10 µg/µL Pepstatin A (or EDTA-free complete protease inhibitor cocktail)) by rocking the lysates for 15 min at 4 °C.
   1. Centrifuge the lysates at 16,000 x g for 10 min at 4 °C.
   2. Collect the supernatant in 1.7 mL microcentrifuge tubes and store at -20 °C until ready to proceed with click reaction.
      NOTE: The protocol can be paused here. Lysates are stable at -20 °C for up to 1 month. However, it is recommended to proceed with the click reaction in a timely manner.
8. Quantify the protein concentrations using an appropriate assay as per the manufacturer's protocol, such as a detergent compatible (DC) assay.

3. Click reaction on cell lysates

1. Prepare the reagents for click chemistry.
   1. Dissolve tris-(benzyltriazolylmethyl) amine (TBTA) in DMSO to 2 mM. Store in small aliquots with desiccant at -20 °C for up to 2-3 months. Best stored under N₂ or Ar.
   2. Dissolve CuSO₄ in ddH₂O water to achieve 50 mM. Store at room temperature for up to 2 months.
   3. Dissolve tris-carboxyethylphosphine (TCEP) in ddH₂O water to 250 mM. Store in darkness at 4 °C and make fresh 50 mM dilutions just prior to the click reaction.
   4. Prepare 2 mM azide in DMSO. Store in small aliquots with desiccant at -20 °C for up to 6 months. Best stored under N₂ or Ar.
      NOTE: It was observed that the products with three or more polyethylene glycols groups worked best with biotin.
2. Bring 50-100 µg of protein lysates in 1.7 mL microcentrifuge tubes to the same volume using the same lysis buffer as above.
   NOTE: Keep the reaction volume as small as possible (20-100 µL).
   1. Add sodium dodecyl sulphate (SDS) to each sample to attain a final 1% concentration.
   2. Prepare a master mix of click reagents so that the final concentrations after addition to the lysates are: 100 µM TBTA (2 mM stock), 1 mM CuSO₄, (50 mM stock) 1 mM TCEP (50 mM stock), and 100 µM azide probe (2 mM stock). Combine stock solutions accordingly (see Table 2).
      NOTE: The order is important. Mix completely after addition of each component.
   3. Add appropriate volumes of the master mix into the lysates. Mix by pipetting up and down.
   4. Incubate for 30 min in the dark in a 37 °C water bath. Agitate/mix occasionally.

Table 2: Click reagent and protein volume ratios. Experimental volumes of the click chemistry reagents and corresponding stock concentrations, in addition to the volumes of protein samples.

4. Click reaction on immunoprecipitated proteins

1. Alternatively, it is possible to perform the click reaction on immunoprecipitated (IP) proteins on or off the beads.
   NOTE: Typically, performing click off the beads leads to the least background and is best when testing new proteins of interest.
   1. Transfect cells for the protein of interest, in this case, wildtype myristoylated C-terminal huntingtin (HTT) fused to GFP (myr-ctHTT-GFP) and ctHTT-GFP with a G2A substitution, using calcium phosphate DNA coprecipitation, as previously described⁴¹.
      1. Plate 2.5 x 10⁵ cells/well in 6-well tissue culture plates and grow overnight to ~70-80% confluency.
      2. Prepare the DNA mix by adding 2.5 µg DNA in 10 µL with 99.75 µL molecular grade H₂O to a 1.7 mL microcentrifuge tube. Then, add 15.25 µL CaCl₂ dropwise to the DNA mix.
      3. Add DNA/CaCl₂ mixture to a separate tube containing 125 µL 2x HEPES buffered saline (HBS, pH 7.0) in a dropwise manner with mixing.
      4. Slowly add the DNA/CaCl₂/HBS mix to cells. After 2-4 h, replace the media and incubate overnight. Proceed with labeling from steps 1.3-2.8.
   2. Prepare equal volumes of 500 µg protein in lysis buffer.
   3. Perform IP by incubating with rabbit anti-GFP for ctHTT-GFP rotating overnight at 4 °C.
   4. Add 15-20 µL protein G beads pre-equilibrated with lysis buffer to each tube and allow it to react end-over-end at 4 °C for 3 h.
   5. Wash the beads with lysis buffer and resuspend in 45 µL 1% SDS 50 mM HEPES buffer.
   6. Heat the beads at 80 °C for 15 min and invert the tubes or agitate approximately every 5 min. Briefly spin the tubes and return to 80 °C.
   7. Collect the supernatant containing the proteins while the samples are still warm.
      NOTE: The protocol can be paused here and the immunoprecipitated samples can be stored at -20 °C or -80 °C, if not used for click chemistry right away.
   8. Allow 43 µL of the supernatant to react with 7 µL master mix of click reagents.
   9. Proceed with the reaction as stated in step 3.2.3.

5. SDS-PAGE and western blotting

1. Stop the reaction and denature by adding 1x sample loading buffer containing 25 mM dithiothreitol (DTT) and heat at 95 °C for 5 min.
   NOTE: Up to 100 mM DTT can be used²⁸. Do not use β-mercaptoethanol with S-acylated proteins as it can hydrolyze the thioester bonds and remove the clicked fatty acid analog.
2. Briefly spin down the samples.
3. Separate the proteins with SDS-PAGE on a polyacrylamide gel, in duplicates.
   NOTE: Two gels are required for the alkaline treatment with KOH to confirm the presence of thioester bonds. If using a fluorescent azide probe, acylation can be detected in-gel or after transfer using the indicated excitation channel.
4. Transfer to polyvinylidene fluoride (PVDF) membranes (activated in methanol and rinsed in ddH₂O – 5 min each) with semi-dry transfer apparatus at 25 V, 1.0 A (constant) for 30 min.
5. After briefly rinsing the membranes with ddH₂O, soak one replicate membrane in 0.1 M KOH in methanol/water (9:1 v/v) and the other in 0.1 M Tris-HCl pH 7.0 in methanol/water (9:1 v/v) as a non-alkaline control for 60 min at room temperature with gentle rocking.
6. Rinse the membranes briefly in ddH2O water, followed by 6 times of 5 min washes in PBS-T (1x PBS, 0.1% Tween 20).
   NOTE: Wash thoroughly.
7. Block the membranes overnight in 5% skim milk blocking buffer (1x PBS, 0.1% Tween20).

6. Perform western blot

1. Where indicated, probe with fluorescently tagged Streptavidin (1:5000) and loading control, anti-GAPDH rhodamine (1:5000) in 5% BSA blocking buffer (0.01% SDS, 1x PBS, 0.1% Tween20) at room temperature for 45-60 min with gentle rocking, in darkness.
   1. Probe the immunoprecipitated protein blot first with primary anti-GFP antibody in 5% skim milk blocking buffer, then with appropriate secondary antibodies in 5% BSA as in step 6.1.
2. Wash the membranes for 5 min in PBS-T (1x PBS, 0.1% Tween20) a total of four repeats and rinse with ddH₂O water before imaging.