Materials and Reagents

1. Escherichia coli (E. coli) Rosetta 2 (DE3) cell line
   
2. pET overexpression system
   
3. His-tag and Ni²⁺-NTA affinity chromatography (HIS-Select Nickel Affinity Gel) (Sigma-Aldrich, catalog number: P6611 )
   
4. AA substrates
   Note: Each enantiomer of the 19 chiral proteinogenic AAs, and the four epimers of hydroxyproline, are prepared in 50 mM HEPES buffer (pH 7.4) with any co-factors (PLP at 20 μM was used in our case.).
   
5. 0.5% solution (w/v, in acetone) of Marfey’s reagent (Sigma-Aldrich, catalog number: 71478 )
   
6. 1 M NaHCO₃
   
7. 2 M HCl
   
8. 2 M NaOH
   
9. HPLC buffer (0.05 M TEAP buffer, pH 3.0) (see Recipes)
   

Equipment

1. Bottle top filter (polystyrene) (Corning, catalog number: 430513 ) (filter used in TEAP buffer preparation)
   
2. Syringe filter (PTFE) (Tisch Scientific, catalog number: SF14466 ) (filter used in HPLC sample preparation)
   
3. Heat block (set at 37 °C for enzyme reactions)
   
4. Heat block (set at 40 °C for derivatization of reaction products)
   
5. 2 ml microfuge tubes (for the reaction, derivatization, and dilution of derivatized products)
   
6. Amber HPLC vials with caps, syringes (1 ml), needles, and filtersfilters (0.22 μm polytetrafluoroethylene, PTFE)
   
7. Waters Nova-Pak (C₁₈ column) (3.9 mm by 150 mm)
   

Software

1. Microsoft Excel

Procedure