Marfey s reagent

A small quantity of 1 was dissolved in 1 mL of 6N HCL and incubated for 6 h at 110 °C using 1.5-mL reaction tubes and a thermoblock. After cooling down to room temperature, the reaction was reduced to dryness by vacuum centrifugation at 40 °C. The dry sample after hydrolysis was re-dissolved in 100 µL of H₂O. The derivatization was carried out by mixing the re-dissolved hydrolystate with 180 µL FDAA in acetone (Marfey’s reagent, Sigma), N_α-(2,4-Dinitro-5-fluorophenyl)-l-alaninamide), and 20 µL 1N NaHCO₃. The reaction was incubated at 40 °C using a thermoblock. After incubation, the reaction was acidified with 30 µL of 1N HCl and diluted with 2.5 mL of methanol. Then, 0.1 mg of l-threonine and d-threonine dissolved in 100 µL water were used to prepare standards of the amino acids using the same derivatization procedure as described for the sample hydrolysate. The standards and sample diluted in methanol were analyzed using UHPLC-MS/MS as described above.

PG (OD₆₀₀ ≈ 1.0) was digested with 20 μg ml⁻¹ pseudoalterin in 20 mM Tris-HCl (pH 9.0) at 25 °C for 24 h. The released amino acids in the hydrolysate were analyzed by an Automatic Amino Acid Analyzer (Hitachi L8900, Japan). The d/l-amino acids and peptides in the hydrolysate were also analyzed by HPLC. The d/l-amino acids in the hydrolysate were derivatized with FDAA (Nα-(2,4-dinitro-5-fluorophenyl)-l-alaninamide, Marfey’s reagent; Sigma) as described by Hess et al.^{48 (link)}. Derivatized amino acids were separated with a linear gradient of formic acid (30 mM, pH 2.6)/acetonitrile on an HPLC with an Inertsil ODS-3 column (250 × 4.6 mm; 5 μm particle size) (Shimadzu, Japan) at a flow rate of 1.5 ml min⁻¹ and detected at 340 nm. Glycine, d-alanine, l-alanine, l-lysine, d-glutamate, AG, AGG, AGGG, AGGGG, AGGGGG, GG, GGG, GGGG, GGGGG, Ae, AeK, AeKa, AeKaA, eK, eKa, eKaA, Ka, KaA, and aA at concentrations of 0.5–4.0 mg ml⁻¹ served as standard solutions.
Peptides AaKAGGGGGA and lactic acid-AeKAGG were synthesized by ChinaPeptides Co., Ltd (China). Each of these peptides (50 μg) was hydrolyzed using 2.5 μg pseudoalterin in 25 μl of 10 mM Tris-HCl (pH 9.0) at 20 °C for 25 h. The hydrolysates were subjected to LC-MS analysis to determine the molecular masses of the released peptides. The sequences of the released peptides were determined by using ExPASy tools.