Figure 1. The two enzymatic reactions of the phenylpropanoid pathway catalyzed by HCT. HCT (AEN02914) converts p-coumaroyl-CoA into p-coumaroyl shikimic acid (first HCT-reaction), which is converted to caffeoyl shikimic acid by C3H (coumarate 3-hydroxylase). HCT converts it further to caffeoyl-CoA (second HCT-reaction).

Materials and Reagents

1. Tris base (2-amino-2-hydroxymethyl-propane-1,3-diol) (Biosolve, catalog number: 77-86-1 )
   
2. Dithiothreitol (DTT) (AG Scientific, catalog number: C-1029 )
   
3. Polyvinylpolypyrrolidone (PVPP) (Sigma-Aldrich, catalog number: P-6755 )
   
4. Glycerol (Sigma-Aldrich, catalog number: G-7893 )
   
5. Complete Mini Protease Inhibitor Cocktail Tablets (Roche, catalog number: 0 4693159001 )
   
6. Bio-Rad Protein Assay (Bio-Rad Laboratories, catalog number: 500-0006 )
   
7. Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A-7906 )
   
8. p-coumaroyl-CoA (TransMIT, catalog number: C030 )
   
9. Shikimic acid (Sigma-Aldrich, catalog number: S-5375 )
   
10. Caffeoyl shikimate (AnalytiCon Discovery GmbH, catalog number: NP-00058 )
    
11. Coenzyme-A hydrate (CoA) (Sigma-Aldrich, catalog number: A-3164 )
    
12. Acetonitrile ULC/MS grade (Biosolve, catalog number: BIO-012041 )
    
13. Formic acid ULC/MS grade (Biosolve, catalog number: BIO-06914131 )
    
14. Liquid chromatography (LC) sample vials (Waters, catalog numbers: 186002639 and 2639531020 ) 
15. Ice
16. Liquid nitrogen
    
17. Protein Extraction Buffer (see Recipes)
    
18. Reaction Mix (see Recipes)
    

Equipment

1. Nunc 96-well microplate without lid and flat bottom wells (Thermo Fisher Scientific, catalog number: 269787 )
   
2. Safe-Lock tubes 2.0 ml (Eppendorf, catalog number: 3706 )
   
3. Stainless steel surgical scalpel blades No.22 (Swann-Morton, catalog number: 0 308 )
   
4. Mortar (150 x 70 mm; 700 ml) (The Morgan Advanced Materials Company plc, catalog number: 12906-6a )
   
5. Pestle (150 x 36 mm) (Haldenwanger, catalog number: 12906-2 )
   
6. Balance (Mettler Toledo, model: XP-105 Delta Range )
   
7. Vortex (IKA, model: MS2 Minishaker L002050 )
   
8. Temperature controlled benchtop microcentrifuge (Eppendorf, model: 5417R )
   
9. Temperature controlled microplate spectrophotometer (Molecular Devices, model: Spectra Max 250 )
   
10. Thermoblock (Eppendorf Thermomixer Compact) or water bath
    
11. UPLC-MS system (In our case: Waters Acquity UPLC system (WATERS) connected to a Thermo LTQ XL mass spectrometer (Thermo Fisher Scientific) or a Synapt HDMS Q-Tof (WATERS). Chromatographic separation was performed on a Waters Acquity BEH RP C18 (2.1 mm x 150 mm, 1.7 μm) column (WATERS))
    
12. Ultrafreezer
    
13. Optional: Qubit 2.0 Fluorometer (Invitrogen)
    

Software

1. XCalibur 2.0 software (Thermo Fisher Scientific, Waltham) was used to acquire, analyze and manage mass spectrometry information

Procedure