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Amino acid - Confirmation of D-amino acids

General description

Twenty common amino acids (except glycine) have one or two chiral centers. Although L-amino acids predominate in mammals, the D-enantiomers also exist and have distinct roles from their L-enantiomers. Instead of direct chiral analysis, the indirect method is also widely used to analyze D-amino acids. The workflow contains two steps: chemical derivatization of D/L-amino acids and then MS analysis coupled to common reverse-phase chromatography. Marfey's reagent is one of the most commonly used ones for enantiomers analysis. The first Marfey's reagent was developed by Marfey in 1984. There are many different Marfey's reagents commerically available. FGCZ currently uses N-(5-Fluoro-2,4-dinitrophenyl)-L-leucinamide (L-FDLA) for D-amino acids analysis and 11 D-amino acids can be separated and detected by our current setup. However, some D-amino acids require different reagents or technology for further confirmation. Please contact us for further information. Proteomics at fgcz.ethz.ch

Workflow for D-amino acid analysis from proteins/peptides

The workflow consists of four major steps:

1. Acid hydrolysis: proteins/peptides are hydrolyzed together with isotope-labeled amino acids under 6M HCl with 0.1% w/v phenol stream at 110°C for 24 hours
2. Labeling: reaction with 5-fluoro-2,4-dinitro phenyl-5-l-leucinamide (L-FDLA). Marfey's reaction
3. LC-QDA analysis: Cortex UPLC reverse-phase separation of the amino acid derivatives
4. Data analysis: Data are processed by TargetLynx™ of MassLynx™ Software (Waters)(MassLynx)
   • Calibration curve establishment: 2.5 uM to 250 uM per amino acid (7 points) by Standard H and isotope-labeled amino acid mixture
   • amino acid quantitifcaiton

Requirements and considerations

• volume: > 5 uL
• minimal amount: 1-5 ug; optimal amount: 10 ug
• dissolve samples in volatile solvents or free of non-volatile buffers, salts, detergents, and primary amine

Reference

1. Bhushan R., Bruckner H. Amino acids 2004, 231-247 https://10.1007/s00726-004-0118-0
2. Yamamoto T, Yaku Keisuke and Nakagawa T 2021, Metabolites, 11, 57-69 https://doi.org/10.3390/metabo11010057