Protocol to identify S-acylated proteins in hippocampal neurons using ω-alkynyl fatty acid analogs and click chemistry (2024)

• •

  A bioorthogonal labeling approach for identifying protein S-acylation

• •

  Protocol for metabolic labeling of neuronal cultures with clickable fatty acids

• •

  Steps for click chemistry to biotinylate S-acylated proteins

• •

  Isolation of S-acylated proteins by affinity purification and hydroxylamine elution

Institutional permissions

If performing experiments using primary rat- or mouse-derived neurons, approval of animal use procedures will be required from the relevant animal ethics committee. All animal use and procedures were approved by the Animal Care Committee of the University of Guelph and followed the guidelines of the Canadian Council on Animal Care (Animal Use Protocol #4478).

25× Roche cOmplete EDTA-free protease inhibitor co*cktail (PIC)

500mL EDTA-free modified RIPA buffer

1L dilution buffer

1L 2% SDS buffer

5mL elution buffer

Fatty acid-free bovine serum albumin (FAF-BSA)

1000× fatty-acid stock solutions

Biotin-azide plus solution

1000× leupeptin solution (Leu)

1000× benzamidine solution (Bz)

100× iodoacetamide solution

10,000× Palmostatin B solution

TBTA solution

THPTA solution

BTTAA solution

Sodium L-ascorbate solution

CuSO₄ solution

Metabolic labeling

Timing: 2–16 h

Metabolic labeling is used to incorporate fatty-acid analogs with a terminal alkyne group that can be used in linking reactions such as click chemistry. By introducing alkynyl-fatty acids, click chemistry can be carried out to isolate S-acylated proteins. There is no interference with cellular processes within cells as alkyne groups are functional groups that do not occur naturally in biological systems. Labeling with fatty acids lacking an alkyne group serves as a negative control for the selectivity of the click reaction and removal of unbound proteins during wash steps.

• 1.

  Saponify fatty acids.

  • a.

    Pre-warm FAF-BSA to 37°C.

  • b.

    In a glass conical vial, add required volume of fatty acid.

    Note: We recommend using 100μM working concentration of fatty acids, therefore, per 2mL of media use 2μL of 100mM fatty acid stock solution.

    Note: Using glass helps to decrease the binding of fatty acids to the surface of the vial.

    Note: The zDHHC family of palmitoylating enzymes have distinct lipid substrate specificities15 so we recommend initially comparing alkynyl-palmitate and alkynyl-stearate for the protein of interest to determine which fatty acid is predominantly used.

    Alternatives: The optimal fatty acid concentration may vary in different cell types as free fatty acids can induce toxicity. We typically use 100μM as toxicity is reduced by the saponification/BSA binding process,8^,9 however, testing concentrations ranging from 10μM to 100μM is advised.

  • c.

    Add an equal volume of 120mM KOH to the fatty acid and gently tap vial to mix, do not pipette.

    Note: The minimum recommended volume for saponification is 4μL, i.e., 2μL of fatty acid plus 2μL of KOH.

  • d.

    Saponify fatty acids by incubating at 70°C for 30 s –3min. Submerge the vials in water in a heat block with 25mm slots or in a water bath.

    Note: Saponification time varies depending on fatty-acid length and volume. Check after 30s by gently tapping the vial to mix. 16-carbon chain fatty acids take approximately 1.25min to saponify, while 18-carbon chain fatty acids take approximately 3min. Fatty acids will change from an opaque white color to clear and colorless when saponified.

    CRITICAL: Proceed to addition of FAF-BSA as soon as the fatty acid-KOH mix turns clear. If it is incubated at 70°C for too long, the DMSO will evaporate, and the fatty acid will precipitate. If this occurs, it cannot be re-saponified, prepare fresh fatty acid-KOH mix.

  • e.

    Add FAF-BSA without letting saponified fatty acids cool down.

    Note: Some precipitation of BSA at this point due to heat will occur, the precipitate should go back into solution during the binding step.

    • i.

      For immortal cells use 1% final FAF-BSA concentration, i.e., for every 2μL of fatty acid and 2μL of KOH add 100μL of FAF-BSA for 2mL culture volume, scaled up or down according to desired culture plate/well size.

    • ii.

      For neuronal cultures use 0.5% final FAF-BSA concentration, i.e., for every 2μL of fatty acid and 2μL of KOH add 50μL of FAF-BSA for 2mL culture volume, scaled up or down according to desired culture plate/well size.

  • f.

    Incubate for 15min at 37°C to bind the fatty acid to BSA.

    Note: 15min is the minimum time. As this solution is stable for extended periods of time, it is acceptable if it remains at 37°C for longer (up to an hour).

• 2.

  Label cells.

  • a.

    Add 100μL (immortal) or 50μL (neuronal cultures) of saponified fatty acid/FAF-BSA mix for every 2mL media to cells dropwise across the surface of the plate.

    Note: To increase uptake of fatty acids when labeling immortalized cells, prior to adding the fatty acids, wash the cells with 1× PBS once, and replace regular media with delipidated media for 30–60min. Delipidated media contains 5% charcoal stripped serum (instead of 10% regular serum), which allows for the depletion of fatty acids from the cells without inducing autophagy and cell death while conserving the expensive charcoal stripped serum. In addition, at this point the plate/well volume can be reduced to further conserve the delipidated media, i.e., for a 10-cm plate use 4mL of delipidated media or for a 60-mm plate use 2mL delipidated media.

    Note: Delipidated media should not be used for primary neuronal cultures that are cultured in Neurobasal media with B27 as a full media change can stress neurons. In addition, Neurobasal media with B27 does not contain saturated fatty acids so no delipidated media is required.

    CRITICAL: Adding fatty acids drop-wise reduces excessive toxicity/cells stress and reduced lipid solubility caused by areas of high fatty acid concentration and ensures even distribution across the entire well/plate.

  • b.

    Incubate 1–16 h.

    Note: Labeling time depends on the experimental set-up and the protein(s) of interest. We recommend first testing a 4–7h. labeling period. A protein with a short S-acylation half-life such as Ras can be labeled in as little as 5min to 1 h, while proteins that are S-acylated more stably and slowly can be labeled for 8–12 h.

    CRITICAL: To avoid fatty acid β-oxidation, we do not recommend labeling cells for more than 16 h.8^,9

• 3.

  Harvest cells.

  • a.

    Following incubation, aspirate the media, wash cells with 1× PBS (immortal cultures) or 1× Recording Buffer+ 1mM MgCl2 (neuronal cultures; 25mM HEPES pH7.4, 120mM NaCl, 5mM KCl, 2mM CaCl₂, 30mM Glucose).

    Note: The buffer used to wash the cells can be any buffer that is commonly used for the given cell type.

  • b.

    Lyse cells in an appropriate volume of ice-cold EDTA-free modified RIPA Buffer, supplemented with PIC and Palmostatin B and collect into 1.5mL centrifuge tubes on ice.

    Note: The volume of lysis buffer will depend on the seeding density and cell type, i.e., for neurons we use 300–500μL per well of a 6-well plate (seeded at a density of 30,000 cells/cm²) or 500–750μL per 60mm dish of HEK293T cells that are 90% confluent.

    Note: Palmostatin B is a potent inhibitor of acyl-protein thioesterases 1 and 2 and it also targets other serine hydrolases.16^,17 We recommend adding it to prevent potential de-acylation due to residual activity of de-S-acylating enzymes in the lysis buffer.

    Alternatives: We recommend testing lysis buffers with different detergents to optimize solubilization of the protein of interest. We have successfully used 2% SDS lysis buffer (2% SDS, 50mM HEPES or Tris pH 7.4) and 1% β-D-maltoside in PBS. Indeed, we were able to successfully detect S-acylation using 2% SDS buffer for a protein that was previously not detectable using the RIPA buffer.

    CRITICAL: Do not harvest in buffers that contain copper chelators, such as EDTA or EGTA, that will interfere with the click reaction.

    Pause point: Samples can be stored at −20°C until ready to proceed.

Click chemistry

Timing: 2 h preparation, 1 h incubation, 16–24h acetone precipitation

Click chemistry relies on the specificity of the copper-catalyzed azide-alkyne bioconjugation reaction. The formation of stable azide-alkyne bonds allows for purification of biotin azide-alkyne-protein conjugates using NeutrAvidin-based capture.

• 4.

  Prepare lysates.

  • a.

    If frozen, thaw cell lysates on ice.

  • b.

    Rotate cell lysates for ½ h at 4°C at 12 rpm on a tube revolver.

  • c.

    Spin down lysates at 17,000×g for 10min at 4°C and carefully remove the supernatant taking care not to disturb the pellet.

Optional: Pass supernatant through a 0.22μm Spin-X centrifuge filter via centrifugation at 8,000×g for 2min at 4°C. This will remove any large insoluble, particulate matter, or aggregates that may non-specifically bind to NeutrAvidin beads increasing the background signal in the negative control. This step, however, can be skipped if interested in detecting S-acylation of aggregation-prone proteins. It may require 2 spins to completely filter supernatant, if the sample still does not pass through filter, increase the speed up to 15000×g and spin for an additional 2min.

Alternatives: Spin half hour, 100000×g to remove all insoluble material.

Optional: Block free thiols that may interfere with the click reaction by adding 1mM iodoacetamide to lysates and incubate for half an hour with rotation at 21°C–24°C.

Pause point: Samples can be frozen and stored at −20°C following protein clearance and blocking.

• 5.

  Prepare inputs.

  • a.

    Run Bio-Rad DC protein assay or other protein quantification assay that is compatible with the lysis buffer.

  • b.

    Dilute 20–50μg of protein in ice-cold EDTA-free modified RIPA buffer (or appropriate lysis buffer used to lyse cells) with PIC to a final volume of 80μL.

  • c.

    Add 20μL of 5× Laemmli Sample Loading Buffer with 5% β-mercaptoethanol (BME; final 1% concentration).

  • d.

    Denature at 50°C for 10min or 95°C for 5min, depending on the protein of interest.

CRITICAL: Transmembrane or aggregation-prone proteins can aggregate if heated at too high a temperature and as a result may not properly resolve on SDS-PAGE gel.

• 6.

  Prepare lysates for click chemistry reaction.

  • a.

    Dilute 150–1000μg of protein lysates in ice-cold EDTA-free modified RIPA buffer (or appropriate lysis buffer used to lyse cells) with PIC according to volumes listed below.

    Note: The required protein amount is dependent on the protein of interest, we recommend first testing 500μg of protein. This amount is sufficient to detect S-acylation of low abundance proteins, while not oversaturating the signal from highly S-acylated proteins. We have successfully performed the click reaction with as low as 150μg of protein.

    Note: The purpose of the chelator ligand is to stabilize and increase the availability of Cu¹⁺. However, due to their different chelating properties and polarity, the chelator ligands are not mutually interchangeable, and a specific chelator ligand may be incompatible with the protein of interest.18 Therefore, we suggest carrying out an initial click chemistry reaction with a direct comparison of all three chelator ligands listed below to determine which yields optimal signal/noise.

    • i.

      TBTA= 455μL.

    • ii.

      THPTA= 415μL.

    • iii.

      BTTAA= 415μL.

• 7.

  Perform click reaction according to tables below (final volume for all is 500μL/reaction).

  • a.

    Add 10μL of biotin-azide plus stock solution to diluted lysates.

  • b.

    Prepare a master mix of TBTA, THPTA, or BTTAA and CuSO_4.

    Note: CuSO₄/chelating agent solution will turn darker blue than CuSO₄ alone

  • c.

    Add required volume of chelator ligand/CuSO₄ master mix to the lysates with biotin-azide plus.

    • i.

      TBTA= 30μL.

    • ii.

      THPTA= 50μL.

    • iii.

      BTTAA= 50μL.

  • d.

    Add required volume of sodium L-ascorbate to lysates to start the reaction.

  • e.

    Incubate 1h at 21°C–24°C in the dark with rotation at 12rpm on a tube revolver.

    	Stock	Working	Volume/reaction
    TBTA	10mM	0.1mM	5μL
    CuSO4	20mM	1mM	25μL
    Na+ L-Ascorbate	100mM	1mM	5μL
    Biotin azide plus	5mM	100μM	10μL

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    	Stock	Working	Volume/reaction
    THPTA	100mM	5mM	25μL
    Na+ L-Ascorbate	300mM	15mM	25μL
    CuSO4	20mM	5mM	25μL
    Biotin azide plus	5mM	100μM	10μL

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    	Stock	Working	Volume/reaction
    BTTAA	100mM	5mM	25μL
    Na+ L-Ascorbate	249mM	12.45mM	25μL
    CuSO4	48mM	2.4mM	25μL
    Biotin azide plus	5mM	100μM	10μL

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• 8.

  Stop the click reaction by adding 10.2μL of 0.5M EDTA, pH= 8.0 (10mM final concentration).

• 9.

  Acetone precipitate protein.

  • a.

    Transfer samples to 15mL conical tubes and add ice-cold acetone to 80% final concentration, i.e., 2mL of 100% acetone for 500μL reaction.

    CRITICAL: Acetone will dissolve polystyrene serological pipettes, use glass serological pipettes only.

  • b.

    Mix thoroughly, incubate at −20°C for 14–16h to fully precipitate proteins.

    Note: Acetone precipitation is required to remove the excess biotin azide plus from the sample.

    Alternatives: Other methods to remove excess biotin azide plus; such as desalting columns, trichloroacetic acid precipitation, or chloroform-methanol precipitation, can be used if they have been previously tested and result in good recovery of the protein of interest.

    Pause point: Precipitated proteins can be kept in acetone for up to 7days.

NeutrAvidin pull-down& hydroxylamine elution

Timing: 2 h preparation, 3 h incubation, 1h elution

Avidin-biotin binding allows for the purification of S-acylated proteins from total lysates. While hydroxylamine cleavage facilitates the specific removal of the S-acylated proteins from NeutrAvidin beads by reducing the thioester bond. Proteins can then be visualized through SDS-PAGE and immunoblot analysis.

• 10.

  Pellet and wash precipitated protein.

  • a.

    Pellet protein precipitate by centrifugation for 5min at 900×g in benchtop centrifuge at 4°C.

  • b.

    Carefully decant supernatant.

  • c.

    Add 6mL chilled acetone to wash pellet and re-spin.

  • d.

    Repeat wash.

  • e.

    Quick spin after final decant.

    Note: Take care not to dislodge the pellet, it is normal for the protein pellets to appear blue.

  • f.

    Remove residual acetone with the gel loading tip.

  • g.

    Allow acetone to completely evaporate but do not over dry as an over dry protein pellet will be difficult to redissolve. A pellet that is sufficiently dry but not over dried should have a slight sheen to it. An over-dry pellet will look matte and paper-like.

  • h.

    Resuspend pellet in 250μL 2% SDS buffer with PIC.

    Note: PIC is added to safeguard against proteases that might not be completely denatured, or proteases introduced from the environment.

  • i.

    Transfer to new 15mL conical tubes and leave on rotator at 21°C–24°C until fully dissolved while preparing the buffers required for next step.

    CRITICAL: Failure to fully dissolve the pellet may result in incomplete recovery of all proteins. The acetone needs to evaporate fully, but do not let the pellets get over dry, as it will make the protein harder to resuspend. Following rotation, pellets can be sonicated if still not fully dissolved.

• 11.

  Purify biotinylated proteins using NeutrAvidin-based capture.

  • a.

    Dilute samples in 4.75mL ice-cold dilution buffer with 150mM NaCl, 1× Leupeptin (Leu), and 1× Benzamidine (Bz).

    Note: Leu and Bz are added to protect against proteases that might have been introduced from the environment as the PIC that was added to the 2% SDS buffer is diluted during this step.

  • b.

    Prepare high capacity NeutrAvidin agarose beads by taking 30μL resuspended bead slurry/sample.

    Note: Use a cut tip when handling beads.

    Alternatives: We recommend using high capacity NeutrAvidin agarose beads rather than regular capacity beads to ensure that the highly abundant proteins do not oversaturate the beads and low-abundance proteins are being fully captured.

  • c.

    Wash beads 3× 500μL dilution duffer with 150mM NaCl, Leu, and Bz. 150mM NaCl is added in fresh to the final concentration required from a stock solution of 5 M, stock dilution buffer is described above in “materials and equipment”.

  • d.

    Following the final wash, resuspend beads in 500 or 1000μL of dilution duffer with 150mM NaCl, Leu, and Bz depending on the volume of beads. Add the dilution buffer until the meniscus is just above the 500 or 1000μL volume marker on the microcentrifuge tube.

    Note: Before aliquoting beads, ensure you have at least the desired volume of beads by taking up with a p1000.

  • e.

    Aliquot an equal volume of NeutrAvidin-agarose to your samples with a cut p200 tip, mixing by gently pipetting up and down between each sample to ensure even distribution of beads.

    Note: The same cut tip, if avoiding touching the insides of the tube, can be used between samples to further reduce pipetting error.

  • f.

    Incubate 3h at 4°C with rotation at 12rpm on a tube revolver.

  • g.

    Prepare wash and elution buffers according to table below during pull-down.

    CRITICAL: Do not place elution buffer on ice as precipitation of buffer may occur. If precipitated, incubate at 37°C for 1–2min and gently vortex before use.

    Buffer	Components	Volume required
    Wash Buffer #1	Dilution buffer with 0.5M NaCl, 1× Leu, 1× Bz	3 washes x 1mL/sample
    Wash Buffer #2	Dilution buffer with 1× Leu, 1× Bz	1 wash x 1mL/sample
    Elution Buffer	Elution buffer (as described in buffer list above) with PIC	40μL/sample

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• 12.

  Elute purified S-acylated proteins.

  • a.

    Pellet beads by centrifugation for 3min, 700×g at 4°C in a benchtop centrifuge.

  • b.

    Aspirate supernatant.

  • c.

    Add 1mL of Wash Buffer #1 to each sample and transfer to a fresh 1.5mL centrifuge tube.

  • d.

    Pulse-spin beads (8000×g) and aspirate supernatant.

  • e.

    Wash pellet twice more with Wash Buffer #1.

  • f.

    Wash 1× times with Wash Buffer #2.

  • g.

    Remove all residual wash buffer with a gel loading tip.

  • h.

    Add 40μL Elution Buffer.

  • i.

    Incubate for 1h at 21°C–24°C with plenty of mixing.

    Note: Do not invert samples. To improve mixing, we suggest using a shaking dry bath set to 1000rpm.

  • j.

    Quick spin to pellet beads

  • k.

    Transfer supernatant to a fresh tube using gel loading tip

    Optional: If protein concentration is very low, resuspend the beads and transfer them toa0.22-micron SpinX column and spin at 7800×g for 5min to collect the entire supernatant.

    Optional: Low-binding microcentrifuge tubes can also be used to collect the eluent if the expected yield is low.

  • l.

    Add 10μL of Laemmli 5× Sample Loading Buffer with 5% BME

  • m.

    Denature protein sample by heating at 50°C for 10min or 95 C for 5min

    Pause point: Samples can be stored at −20°C until ready to proceed with western blotting.

• 13.

  Proceed with SDS-PAGE, western, and immunoblot, using any protocol of choice.

Note: Hydroxylamine elution buffer will not interfere with Tris-Glycine gel chemistry, howeverwe have not tested resolving the samples eluted in hydroxylamine on any other gel chemistry.

Problem 1

Click reaction and pull-down fail to yield a detectable amount of S-acylated protein of interest.

Potential solution 1

Before proceeding to other troubleshooting steps, test the performance of the click assay by probing for well S-acylated proteins that are expressed in the cells of interest, such as calnexin (runs at ∼90kDa), N-Ras/H-Ras (run at ∼25kDa), SNAP-25 (runs at ∼25kDa), PSD-95 (runs at ∼95kDa), or GAP-43 (runs at ∼45kDa).

Problem 2

The background signal in the control sample labeled with fatty acid without an alkyne group is high.

Potential solution 2

This issue could occur for several reasons. First, DMSO-soluble chelator ligands can cause protein precipitation due to the high final DMSO concentration in the reaction (click chemistry step #7). In this case, other water-soluble chelator ligands should be tested. Second, the protein may be prone to non-specific binding to the NeutrAvidin beads during pull-down and/or not being completely removed during the wash steps ( step #12). In this case, beads can be first blocked in 1% BSA dissolved in PBS prior to pull-down step. Wash buffers can also be supplemented with a up to 0.5% SDS to increase the stringency of the wash steps and/or wash step times can be extended.

Problem 3

Following acetone precipitation and 16- to 24-h incubation at −20°C ( step #10) no protein pellet or smaller-than- expected pellet is observed.

Potential solution 3

Ensure that the amount of 100% acetone added yields 80% final concentration. Thoroughly mix samples following acetone addition. If still no precipitate is observed, double-check the calculations for the protein amount used in the reaction. We have successfully used acetone precipitation for as little as 150μg of protein in 500μL.

Problem 4

Click chemistry could fail with proteins that contain free thiols due to strong copper-thiol interaction. This will limit the copper availability in solution for the click reaction.

Potential solution 4

Prior to performing click reaction (i.e., right after click chemistry step #4 c.), 1mM iodoacetamide should be added to lysates for half an hour to block the free thiol groups which will increasing copper availability in the reaction.

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Shaun Sanders (ac.hpleugou@30ednass).

Technical contact

Technical questions should be directed to and will be fulfilled by the technical contact, Charlotte Townsend (ac.hpleugou@10snwotc).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate any unique datasets or codes.

This study was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC; RGPIN-2021-02547). We thank all members of the Sanders’ Lab as well as Dr. Dale Martin (University of Waterloo), who assisted with helpful discussions during the development of this protocol. We would also like to acknowledge that the University of Guelph resides on the Dish with One Spoon territory and treaty lands of the Mississaugas of the Credit and the traditional lands of the Hodinöhsö:ni and Anishinaabeg people. We offer respect and gratitude to all First Nations, Métis, and Inuit people who now call these lands home.

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