Identification of Acetylation Sites of Fatty Acid Synthase (FASN) by Mass Spectrometry and FASN Activity Assay

Materials and reagents

Flies

Fly lines used in this study: yw^R (a gift from Eric Rulifson); yw^R; FASN^Flag (Miao et al., 2022). Flies were maintained at 25 °C, 60% relative humidity, and a 12:12 h light/dark cycle. Adults and larvae are reared on standard cornmeal and yeast-based food.

Reagents

1. Pierce^TM IP lysis buffer (Thermo Scientific^TM, catalog number: 87787)

2. Protease inhibitor cocktail (PIC, 100×) (Sigma-Aldrich, catalog number: P8340)

3. PMSF protease inhibitor (Fisher Scientific, Thermo Scientific^TM, catalog number: PI36978)

4. Sodium butyrate (VWR, Thermo Scientific^TM, catalog number: AAAA11079-06)

5. Nicotinamide (NAM) (Sigma-Aldrich, catalog number: 72340)

6. Pierce^TM BCA Protein Assay kit (Thermo Scientific^TM, catalog number: 23225)

7. SureBeads^TM Protein G magnetic beads (Bio-Rad, catalog number: 1614023)

8. 2× Laemmli sample buffer (Bio-Rad, catalog number: 1610737)

9. 10× Premixed electrophoresis buffer (Bio-Rad, catalog number: 1610732)

10. 4%–20% Mini-PROTEAN^® TGX^TM Precast protein gels (Bio-Rad, catalog number: 4561093)

11. Sucrose (Sigma-Aldrich, catalog number: S9378)

12. Gibco^TM HEPES (1 M) (Fisher Scientific, catalog number: 15-630-080)

13. Magnesium chloride (MgCl₂) (Fisher Scientific, Thermo Scientific Chemicals, catalog number: AC223210010)

14. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632)

15. UltraPure^TM 0.5 M EDTA, pH 8.0 (Fisher Scientific, Invitrogen^TM, catalog number: 15-575-020)

16. Pierce^TM Coomassie (Bradford) Protein Assay kit (Thermo Scientific^TM, catalog number: 23200)

17. Monoclonal ANTI-FLAG^® M2 antibody (Sigma-Aldrich, catalog number: F1804-50UG)

1. 100 mM PMSF

2. 5 M Sodium Butyrate

3. 1 M NAM

4. 1 M Sucrose

5. 1 M MgCl₂

6. 1 M DTT

7. 1 M Dipotassium phosphate (K₂HPO₄)

8. 1 M Potassium dihydrogen phosphate (KH₂PO₄)

9. 24 mM NADPH

10. 6.2 mM Acetyl-CoA

11. 5.8 mM Malonyl-CoA

12. Protein lysis buffer for immunoprecipitation and electrophoresis (lysis buffer 1) (see Recipes)

13. Protein denature buffer for protein elution and electrophoresis (denature buffer) (see Recipes)

14. Electrophoresis running buffer (see Recipes)

15. Protein lysis buffer for FASN activity assay (lysis buffer 2) (see Recipes)

16. 200 mM potassium phosphate buffer (PPB), pH 6.6 (see Recipes)

17. FASN reaction buffer (see Recipes)

Recipes

1. Protein lysis buffer for immunoprecipitation and electrophoresis (lysis buffer 1)

   6–7 mL of lysis buffer 1 will be used for lysis and washing steps; calculate the total volume according to number of samples and make the buffer freshly.

   Pierce^TM IP lysis buffer supplied with 1× PIC, 1 mM PMSF, 50 mM sodium butyrate, and 50 mM NAM




2. Protein denature buffer for protein elution and electrophoresis (denature buffer)

   Note: Make fresh.

   950 μL of 2× Laemmli sample buffer

   50 μL of BME

   1 mL of lysis buffer 1




3. Electrophoresis running buffer

   Note: Can be stored at room temperature.

   10× Premixed electrophoresis buffer following dilution to 1× with water




4. Protein lysis buffer for FASN activity assay (lysis buffer 2)

   300 μL of lysis buffer 2 will be used for the lysis step; calculate the total volume according to number of samples and make the buffer freshly.

   250 mM sucrose

   20 mM HEPES Buffer, pH 7.2

   2 mM MgCl₂

   1 mM DTT

   1 mM EDTA

   1× PIC




5. 200 mM potassium phosphate buffer (PPB), pH 6.6

   Note: Stable at 4 °C for two months.

   0.76 mL of 1 M K₂HPO₄

   1.24 mL of 1 M KH₂PO₄

   Bring volume to 10 mL with distilled water




6. FASN reaction buffer

   200 μL of the reaction buffer will be added to each sample/standard; calculate the volume according to number of standards and samples and make the buffer freshly.

   200 mM PPB, pH 6.6

   1 mM DTT

   1 mM EDTA

   0.24 mM NADPH

   0.031 mM Acetyl-CoA

Laboratory supplies

1. Pellet Pestle^TM cordless motor (Fisher Scientific, catalog number: 12-141-361)

2. Magnetic bead rack (Bio-Rad, catalog number: 1614916)

3. 1.7 mL low-binding microcentrifuge tube (Fisher Scientific, catalog number: 07-200-184)

Equipment

1. Sonifier (Branson, model: SFX150)

2. Microcentrifuge (Sorvall^TM Legend^TM Micro 21)

3. Digital dry baths/block heaters (Fisher Scientific, catalog number: 88860103)

4. Microplate spectrophotometer (BioTek Epoch 2)

5. ChemiDoc MP Imaging System (Bio-Rad)

Procedure

Part I: Identification of acetylation sites by mass spectrometry

1. Protein extraction

   1. Collect 30–50 mg of third-instar larvae (L3) of FASN^Flag flies with a Flag knock-in at FASN locus in a microcentrifuge tube. Samples can be flash-frozen in liquid nitrogen and stored at -80 °C.

   2. Make lysis buffer 1 right before protein extraction.

   3. Add 300 μL of lysis buffer 1 to each tube. Homogenize larvae with a pestle gun on ice until larvae are shattered.

   4. Sonicate for 10 s at 10–20 amplitude.

   5. Bring volume to 800 μL with lysis buffer 1. Incubate samples on ice for 20 min.

   6. Spin at 20,000× g for 30 min at 4 °C.

   7. Transfer the supernatant to a new tube. Repeat the centrifugation step twice or until the supernatant is clear.

   8. Pipette 5 μL of lysate to a new tube. Dilute ten times with lysis buffer 1. The protein concentration of each sample is determined by Pierce^TM BCA Protein Assay according to the manufacturer’s protocol.

   9. Dilute protein to an equal amount according to the Pierce^TM BCA Protein Assay. The optimal amount of total protein for FASN pull down assay is approximately 1 mg. Bring volume to 1 mL with lysis buffer 1.

   10. Pause step: Proteins can be snap frozen in liquid nitrogen and stored at -80 °C.




2. Immunoprecipitation

   1. Add 25 μL of SureBeads^TM Protein G magnetic beads to a clean microcentrifuge tube.

   2. Wash the beads three times with 200 μL of lysis buffer 1: vortex for 5 s to resuspend the beads and centrifuge at 10,000× g for 1 min. Collect beads using a magnetic bead rack and remove the buffer by pipetting.

   3. Add protein lysate from Step A directly to the beads. Incubate at 4 °C on a rotor for 30 min to remove non-specific bindings.

   4. Briefly spin down the bead-lysate mixture. Collect beads using a magnetic bead rack and transfer lysate to a fresh microcentrifuge tube.

   5. Add 10 μL of ANTI-FLAG^® M2 antibody (anti-FLAG) to 1 mL of protein lysate (1:100 dilution).

   6. Incubate antibody-lysate mixture on a rotor at 4 °C overnight.

   7. Add 100 μL of SureBeads^TM Protein G magnetic beads to a clean microcentrifuge tube. Wash the beads three times with 500 μL of lysis buffer 1 as described above.

   8. Add the antibody-lysate mixture to the beads. Vortex briefly to resuspend the beads.

   9. Incubate the antibody-lysate-beads mixture on a rotor at 4 °C for 3 h.

   10. Collect beads using a magnetic bead rack and discard the liquid by pipetting.

   11. Wash the beads three times with 1 mL of lysis buffer 1. Rotate at 4 °C for 10 min between each wash.

   12. Collect beads using a magnetic bead rack and carefully remove all liquid. Add 30 μL of denature buffer. Resuspend the beads by gently tapping the tube.

   13. Heat samples at 95 °C for 5 min to elute proteins from the beads, gently tapping the tube at the halfway point.

   14. Briefly spin samples and place the tube in a magnetic bead rack. Transfer elution to a new microcentrifuge tube.

   15. Pause step: Eluted and denatured proteins can be stored at -20 °C or lower.




3. Electrophoresis and Coomassie Blue staining

   1. Load 30 µL of protein samples from Procedure B on a 4%–20% Mini-PROTEAN^® TGX^TM Precast protein gel for electrophoresis.

   2. Apply Coomassie Blue stain to the protein gel according to the manufacturer’s protocol to visualize the protein.

   3. Cut protein bands with approximately 250 kDa molecular weight; the gel slices can be temporally stored in a microcentrifuge tube with distilled water at 4 °C (Figure 1).

      
      

      
      Figure 1. Protein electrophoresis gel showing immunoprecipitated fatty acid synthase (FASN) protein after Coomassie Blue stain. Protein bands at approximately 250 kDa were cut for further analysis.

      
      

   4. Samples are shipped to a Mass Spectrometry Facility for in-gel digestion, desalting, lyophilization, and MS analysis to detect acetylation sites.




4. Acetylated site identification by mass spectrometry and data validation

   1. Proteomics analysis to identify acetylated lysine residues is normally performed by the Mass Spectrometry Facility. Raw data can also be acquired and processed through proteomics data analysis platforms (e.g., MaxQuant) if further analysis is desired. The use of MaxQuant has been well summarized before (Tyanova et al., 2016).

   2. MS-based analysis has identified eight acetylated lysine residues of FASN that are immunoprecipitated from L3 stage larvae, and the results are compared with previously identified acetylation sites of FASN, which can be acquired from open-access databases such as Protein Lysine Modification Database (PLMD) (Xu et al., 2017) and PhosphoSitePlus^® PTM Resource (Hornbeck et al., 2014) (Table 1).

      
      

      Table 1. List of the eight acetylated lysine residues of fly FASN identified by mass spectrometry

      Ac-Lysine identified in this study	Ac-Lysine at the same position identified previously	Peptide sequence
      K193	None	DRIGKLKDSDLENF
      K333	None	GAGLILKPTMSLQF
      K407	None	GITYPIGKMQNRLIR
      K813	K813 (Drosophila melanogaster, from a different fly study)	NAEGVFAKVNSSGY
      	K673 (Homo sapiens)	RKEGVFAKVRTGGM
      	K673 (Mus musculus)	KQEGVFAKVRTGGL
      	K673 (Rattus norvegicus)	KQEGVFAKVRTGGL
      K926	K926 (Drosophila melanogaster, from a different fly study)	ATNLSLVKGHENNV
      	K786 (Homo sapiens)	CTIIPLMKDHRDNL
      	K786 (Mus musculus)	CTIIPLMKDHKDNL
      	K786 (Rattus norvegicus)	CTIIPLMKDHKDNL
      K1535	None	LISNVLKNGAWGT
      K1800	K1771 (Homo sapiens)	GRFLEIGKDLSQNH
      	K1764 (Mus musculus)	GRFLEIGKDLSNNH
      	K1765 (Rattus norvegicus)	GRFLEIGKDLSNNH
      K2466	K2426 (Homo sapiens)	AARSFYYKRAAEQY
      	K2419 (Mus musculus)	AAVSFYHKRAADQY

   
   

   3. Acetylation of the lysine residue K813 is further validated using an Ac-K813 specific antibody.

   1. To verify the acetylation of K813 residue identified from MS, we first generated a rabbit polyclonal antibody against synthetic peptides with acetylated K813 (Yenzym antibodies, CA, USA). The peptide sequence is NAEGVFA-acK-AVNSSG. which covers amino acids 806–819 of the MAT domain of the fly FASN protein.

   2. We then expressed recombinant KS-MAT didomain of FASN protein using E. coli BL21 (DE3) expression system for the use of acetylation validation experiment.

   3. To validate the acetylation of K813 of the KS-MAT recombinant protein, we incubated recombinant proteins with different amounts of acetyl-CoA for 1 h and checked the acetylation of K813 by western blot analysis and Ac-K813 antibody. Interestingly, K813 was rapidly acetylated by acetyl-CoA in a dosage-dependent manner (Figure 2).

   
   

   
   Figure 2. Acetylation of K813 residue of the recombinant fatty acid synthase (FASN) protein (KS-MAT domain) was detected after Acetyl-CoA (AcCoA) treatment. Acetyl-K813 antibody was used in the western blotting analyses.

Part II: FASN activity assay

1. Protein extraction

   1. Collect 20–30 mg of L3 larvae of yw^R flies in a microcentrifuge tube. Samples can be flash-frozen in liquid nitrogen and stored at -80 °C.

   2. Add 300 μL of lysis buffer 2 to each tube. Homogenize larvae with a pestle gun on ice until larvae are shattered.

   3. Sonicate for 10 s at 10–20 amplitude.

   4. Centrifuge at 3,500× g for 10 min at 4 °C.

   5. Transfer lysate to a new tube and centrifuge again at 21,000× g for 20 min at 4 °C. Repeat twice.

   6. Pipette 5 μL of lysate to a new tube. Dilute ten times with lysis buffer 2. The protein concentration of each sample (mg/mL) is determined by Pierce Coomassie Bradford assay according to the manufacturer’s protocol.

   7. Pause step: Proteins can be temporarily stored at -80 °C.




2. FASN activity assay

   1. To generate NADPH standard curve, measure the absorbance of different dilutions of NADPH: dilute 24 mM NADPH stock solution with distilled water to prepare 12, 6, 3, 1.5, and 0.75 mM standard solutions. Add 200 μL of distilled water to the wells of a 96-well plate and 2 μL of different dilutions of NADPH to generate 0.24, 0.12, 0.06, 0.03, 0.015, and 0.0075 mM/well standards. Read at A_340nm on a plate reader.

   2. On the same 96-well plate, add 20 μL of fly protein extracts and 200 μL of freshly made FASN reaction buffer.

   3. Read plates at A_340nm at 37 °C for 3 min (1 min intervals) to measure background NADPH oxidation.

   4. Add 2 μL of 5.8 mM Malonyl-CoA (final concentration: 58 μM) to each well.

   5. Read at A_340nm at 37 °C for 15 min (1 min interval) to determine FASN-dependent oxidation of NADPH.




3. Calculation

   1. Use the values obtained from the appropriate NADPH standards to plot a standard curve.

   2. The rates of A_340nm change are corrected for the background rate of NADPH oxidation. FASN activity is normalized against protein amount and calculated in nmol NADPH oxidized min^-1 mg protein^-1.

      1. Calculate the average reduction of OD readings within 15 min: Rate 1 = (OD_0 min - OD_15 min)/15. Calculate the rate of OD change of background readings within 3 min: Rate 2 = (OD_0 min - OD_3 min)/3. Correct the rate of A_340nm change: Rate = Rate 1 - Rate 2.

      2. Convert the corrected OD reduction rate (Rate) to change of NADPH concentration (mM) per minute according to the standard curve (C). Calculate the reduction of NADPH amount per min in 200 μL reaction: C × 200 (nmol/min).

      3. Calculate the amount of protein added to each well (mg): Protein concentration (mg/mL) × 0.02 mL.

      4. Normalize reduction of NADPH against protein amount (nmol/min/mg of protein).

      5. In Figure 3, we show that FASN activity decreases in the fly larvae with a specific acetylation-deficiency mutant (FASN^K813R) where the lysine residue at 813 position was mutated to arginine.

      
      

      
      Figure 3. Fatty acid synthase (FASN) enzymatic activity of wild-type fly larvae (WT) and two FASN acetylation-deficiency mutants (FASN^K813R, FASN^K926R). FASN activity was determined by oxidated NADPH per minute per milligram of protein. Fold-change of FASN activity is presented. Data are presented as mean ± SD. One-way ANOVA; ***p-value < 0.001; ns. not significant.

Acknowledgments

We thank Ross Tomaino from Harvard Medical School Taplin Mass Spectrometry Facility for mass spectrometry analysis. We thank Dr. Justin Walley for advising on sample preparation for identification of acetylation sites. Graphical Overview was created with BioRender.com. This work was supported by NSF CAREER 2046984 and NIH R01AG058741 to H.B.

Competing interests

The authors declare no conflicts of interest.

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