Phosphoproteomic Analysis and Organotypic Cultures for the Study of Signaling Pathways

Materials and reagents

Biological materials

For organotypic tissue culture

1. MRC-5 (ATCC, catalog number: CCL-171)

2. N/TERT-1 (James G. Rheinwald, Harvard Institute of Medicine)

   
   

For proteomics

1. Amine (NH2) functional microparticles, MagReSyn^® Amine [ReSyn Biosciences (Pty) Ltd., South Africa]

2. Hydroxyl terminated beads, MagReSyn^® Hydroxyl [ReSyn Biosciences (Pty) Ltd., South Africa]

3. Magnetic microparticles with chelated Ti4 + metal ions for highly specific phosphopeptide enrichment, MagReSyn^® Ti-IMAC HP [ReSyn Biosciences (Pty) Ltd., South Africa]

   
   

Reagents

For organotypic tissue culture

1. Penicillin/streptomycin (Thermo Fisher, Gibco, catalog number: 151401)

2. DMEM (Thermo Fisher, Gibco, catalog number: 41966029)

3. Keratinocyte-SFM (Thermo Fisher, Gibco, catalog number: 17005034)

4. TrypLE (Thermo Fisher, Gibco, catalog number: 12605028)

5. Bovine pituitary extract (BPE) (Thermo Fisher, Gibco, catalog number: 13028014)

6. Fetal calf serum (FCS) (HyClone, catalog number: SH30071.03HI)

7. EGF (Thermo Fisher, Gibco, catalog number: PHG0314)

8. Insulin (Sigma-Aldrich, catalog number: 11070-73-8)

9. Hydrocortisone (Sigma-Aldrich, catalog number: H0888)

10. Triiodothyronine (T3) (Sigma-Aldrich, catalog number: T2752)

11. L-Glutamine (Thermo Fisher, Gibco, catalog number: A2916801)

12. Hams F12 (Thermo Fisher, Gibco, catalog number: 11765-054)

13. Rat tail collagen I (made in own lab [12])

14. Adenine (Sigma-Aldrich, catalog number: A8626)

15. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: H1758)

16. Dulbecco’s phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: D5652)

17. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7979)

18. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 655104)

19. Calcium chloride (CaCl₂·2H₂O) (Sigma-Aldrich, catalog number: C7902)

20. Gentamycin sulphate (Sigma-Aldrich, catalog number: G1264)

21. Cholera enterotoxin (Sigma-Aldrich, catalog number: C8180)

22. 10× minimum essential media (10× MEM) (Thermo Fisher, Gibco, catalog number: 11430-030)

23. Sodium bicarbonate (Thermo Fisher, Gibco, catalog number: 15750037)

24. Tissue-Tek^® (SAKURA, catalog number: 4583)

25. Formalin solution neutral buffered, 10% (Sigma-Aldrich, catalog number: HT501128)

26. Sucrose (Sigma-Aldrich, catalog number: S0389)

27. Isopentane (Sigma-Aldrich, catalog number: PHR1661)

    
    

For proteomics

1. Lysis buffer, composed of 2% sodium dodecyl sulfate (SDS), 5 mM tris(2-carboxyethyl)phosphine (TCEP), 5.5 mM chloroacetamide (CAA), and 100 mM Tris pH 8.5; used for cell lysis

2. Acetonitrile (ACN), LC grade (Supelco^TM Analytical, catalog number: 75-05-8), used in various concentrations throughout the protocol, namely 70% for dilution of lysates, 95% for washing proteins, and 40% and 60% for elution on SepPak. ACN is also used for TMT-labeling

3. Ethanol (anhydrous) (Sigma-Aldrich, catalog number: 459836), used in 70% concentration for washing proteins after aggregation

4. 50 mM ammonium bicarbonate (ABC) (Sigma-Aldrich, catalog number: 09830), used for protein digestion and for resuspending TMT-labeled peptides

5. 50% trifluoroacetic acid (TFA), HPLC (Supelco^TM Analytical, catalog number: TX1276), used for acidification of samples post digestion

6. 1 M TEAB, pH 8 (Thermo Scientific^TM, catalog number: 90114), used to achieve a final concentration of 100 mM in TMT-labeling

7. 50% hydroxylamine (Thermo Scientific^TM, catalog number: 90115), used to quench the TMT-labeling reaction

8. 0.1% formic acid (v/v) in water (LC–MS grade) (Thermo Scientific^TM, catalog number: 85170), used for resuspension of each TMT-labeled sample before pooling

9. Loading buffer, composed of 80% ACN, 5% TFA, and 1 M glycolic acid (Sigma-Aldrich, catalog number: 8.04104); used for resuspending peptides for phosphopeptide enrichment

10. 1% ammonia (ammonia solution 25%) (Supelco, catalog number: 105428), used for elution of phosphopeptides

11. 5 mM ABC (buffer A) and 100% ACN (buffer B), used in high pH reversed-phase peptide fractionation

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

13. TMTpro^TM 16plex label reagent set (Thermo Scientific^TM, catalog number: A44520)

14. LysC, Lysyl endopeptidase (Fujifilm Wako, catalog number: 129-02541)

15. Trypsin, Sequencing grade modified trypsin (Promega, catalog number: V511X)

16. Sep-Pak C18 1 cc Vac Cartridge (Waters, catalog number: WAT054955)

17. Acquity CSH C18 1.7 μm, 1 mm × 150 mm column (Waters, catalog number: 186006935)

    
    

Solutions

1. Adenine (see Recipes)

2. Cholera enterotoxin (CT) (see Recipes)

3. Hydrocortisone (HC) (see Recipes)

4. Epidermal growth factor (EGF) (see Recipes)

5. Triiodothyronine (T3) (see Recipes)

6. Calcium chloride (CaCl₂) (see Recipes)

7. Insulin (1,000× for 5 µg/mL) (see Recipes)

8. Sodium bicarbonate (71.2 mg/mL milli q water) (see Recipes)

9. Keratinocyte serum free medium (K-SFM) complete (see Recipes)

10. Organotypic culture medium (see Recipes)

11. DMEM 10% FBS (DMEM-10) (see Recipes)

    
    

Recipes

For organotypic tissue culture

1. Adenine

   Prepare 2.4 mg/mL of adenine in HCl (this is a 100× stock):

   1. Dissolve 243 mg of adenine in 100 mL of 0.05 M HCl.

   2. Stir for approximately 1 h at room temperature (RT).

   3. Filter sterilize with 0.2 μm filters.

   4. Divide into 5 mL aliquots.

   5. Store at -20 °C.

      
      

2. Cholera enterotoxin (CT)

   Prepare 10 µM solution of cholera enterotoxin (this is 1,000× stock solution for 100 pM):

   1. Add 1.18 mL of Milli-Q water to a 1 mg vial of cholera enterotoxin to prepare a 10^-5 M solution.

   2. Dilute this in 110 mL of PBS.

   3. Filter sterilize with 0.2 µm filters.

   4. Aliquot 2 mL per cryovial.

   5. Store at -20 °C.

      Note: MW_CT = 84.000 Da

      
      

3. Hydrocortisone (HC)

   Prepare 0.2 mg/mL hydrocortisone (this is a 500× stock for 0.4 µg/mL):

   1. Dissolve a 25 mg vial in 5 mL of 100% ethanol. This solution is 5 mg/mL, which is 12.5× for 0.4 µg/mL. This can be stored at -20 °C indefinitely.

   2. Add 2 mL of this concentrated stock to 48 mL of sterile PBS + 0.1% BSA.

   3. Filter-sterilize with 0.2-µm filters.

   4. Aliquot 2 mL into cryovials.

   5. Store at -20 °C.

      
      

4. Epidermal growth factor (EGF)

   Prepare 10 µg/mL of EGF (this is a 50,000× stock for 0.2 ng/mL):

   1. Dissolve 100 µg of EGF in 10 mL of PBS + 0.1% BSA.

   2. Filter sterilize with 0.2 µm filters.

   3. Aliquot 1 mL into cryovials.

   4. Store at -20 °C.

      
      

5. Triiodothyronine (T3)

   Prepare 20 nM (2 × 10^-8 M) of T3 [this is a 1,000× stock for 20 pM (2 × 10^-11 M)]:

   1. Measure 6.8 mg of T3 and dissolve in 7.5 mL of 0.02N NaOH.

   2. Add 42.5 mL PBS (this is 100,000× for 20 pM T3).

   3. Store at -20 °C in 10 mL aliquots.

   4. To make the 1000× stock, dilute 0.4 mL of the 100,000× 1:100 into 39.6 mL of PBS.

   5. Filter sterilize with 0.2 µm filters.

   6. Divide into 2 mL aliquots.

   7. Store at -20 °C.

      
      

6. Calcium chloride (CaCl₂)

   Prepare using calcium dihydrate CaCl₂·2H₂O (this is a 1,000× stock for 0.3 mM):

   1. Dissolve 4.4 g of CaCl₂·2H₂O in 100 mL of Milli Q water.

   2. Stir with magnetic bar to dissolve completely.

   3. Filter sterilize with 0.2 µm filters.

   4. Aliquot 2 mL per cryovial.

   5. Store at -20 °C.

      
      

7. Insulin (1,000× for 5 µg/mL)

   Use insulin from bovine pancreas.

   1. Dissolve 100 mg of insulin in 20 mL of 0.005 M HCl in a 50 mL blue-capped tube.

   2. Filter sterilize through a celluloseacetate, lowprotein binding 0.2 µM filter.

   3. Aliquot 500 µL per tube.

   4. Store at -20 °C.

      
      

8. Sodium bicarbonate (71.2 mg/mL milliQ water)

   
   

For organotypic cultures

1. Keratinocyte serum free medium (K-SFM) complete

   Reagent	Final concentration	Quantity
   Keratinocyte-SFM	n/a	500 mL
   CaCl2 (1,000×)	0.3 mM	500 µL
   BPE (16.8 mg/mL)	25 µg/mL	750 µL
   EGF (10 µg/mL)	0.2 ng/mL	10 µL
   Total	n/a	501.26 mL

   Optional: Penicillin/Streptomycin can be added to K-SFM as well (100 U/mL).

   
   

2. Organotypic culture medium

   Reagent	Final concentration	Quantity
   DMEM	n/a	375 mL
   F12	n/a	125 mL
   Pen/Strep	100 U/mL	5.14 mL
   Adenine (2.4 mg/mL stock)	24 µg/mL	5.14 mL
   HC (0.2 mg/mL stock)	0.4 µg/mL	1 mL
   CT (10 µM stock)	100 pM	0.514 mL
   T3 (1,000×)	20 pM	0.514 mL
   Insulin (1,000×)	5 µg/mL	500 µL
   FCS	0.3%	1.54 mL
   Total		514.3 mL

   
   

3. DMEM 10% FBS (DMEM-10)

   Reagent	Final concentration	Quantity
   DMEM	n/a	445 mL
   FBS	10%	50 mL
   Pen/Strep	100 U/mL	5 mL
   Total		500 mL

   
   

Laboratory supplies

For organotypic tissue culture

1. Deep-well plates (Corning, catalog number: 355467)

2. Cell culture insert, PET membrane, 3.0 µm pore size (Corning, catalog number: 353092)

3. Airlift pads (cut in circles and autoclaved) (Whatman, GE Healthcare, catalog number: 10382461)

4. Cell culture Petri dishes or flasks from any brand (we use NUNC, Falcon, and Corning)

Equipment

For organotypic tissue culture

1. CO₂ incubator at 37 °C (any brand can be used)

2. Centrifuge (any brand, for 15 mL tubes and a minimum of 100 g)

3. Freezers (-20 °C and -80 °C)

   
   

Equipment and software for proteomics

1. KingFisher^TM Flex purification system (Thermo Scientific, catalog number: 5400610)

2. SpeedVac (Eppendor, model: Vacufuge^TM Concentrator)

3. Sonicator (Fisherbrand, model: 120 Sonic Dismembrator)

4. Spectrophotometer (Thermo Scientific, model: NanoDrop^TM 2000 C)

5. HPLC system [Thermo Scientific, model: UltiMate^TM 3000 Standard (SD)]

6. Evosep ONE (Evosep)

7. Mass spectrometer (Thermo Scientific, model: Orbitrap Exploris^TM 480)

8. Proteome Discoverer software v2.4 (Thermo Scientific)

9. Chromeleon software v7.2 (Thermo Fisher Scientific)

Procedure