Chiral amino acid analysis

Microwave assisted vapor phase acid hydrolysis of peptides was performed in a CEM Discover 3000 Microwave System (CEM). Peptides were hydrolyzed in a sealed chamber with 6 N deuterium chloride (DCl) at 150 °C for 30 min under anaerobic conditions. Hydrolysates were derivatized with Marfey’s reagent (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide, FDAA) (Thermo-Fisher Scientific) to enhance separation of L-/D-amino acids. Hydrolysates were reconstituted in 25 µL of 0.5 M NaHCO3, combined with 20 µL of 1 mg/mL FDAA in MeCN, and incubated at 60 °C for 3 h. Derivatized amino acids from each peptide sample were analyzed using a LC-MS/MS-MRM system consisting of an Advance Ultra-High Pressure Liquid Chromatography (UHPLC) (Bruker) system coupled to an EVOQ Elite Triple-Quadrupole MS (Bruker) with a Kinetex 2.6 µm Phenyl-Hexyl 100 Å LC column (100 × 2.1 mm) (Phenomenex). Gradient elution was performed with binary solvents (Solvent A: 25 mM ammonium formate, Solvent B: MeOH) at 300 µL/min. The gradient was 5% B for 2 min, 5–15% B for 5 min, 15–60% B for 5 min, 60% B for 3 min, 60–100% B for 3 min, 100% B for 3 min, 100–5% B for 1 min, 5% B for 2 min. Data Review (Bruker) was used to analyze the MRM data.