Quantitative analysis of peptides with NMR spectroscopy

The determination of peptide concentration with H-1 nuclear magnetic resonance (NMR) spectroscopy using an internal standard or an external standard in a sealed glass capillary was investigated for three tyrosine-containing tripeptides. Trimethylsilylpropionic acid (TSP) and maleic acid were tested as external standards for quantitation by proton NR IR. Although comparable results were obtained for either standard, the performance of maleic acid was found to be superior because of its better long-term stability in the sealed capillary. Loss of TSP from solution occurred over time due to adsorption onto the walls of the capillary, necessitating frequent recalibration against the primary standard, potassium acid phthalate (KHP). The peptide contents of solid peptides determined with H-1 NMR are compared with those obtained from ultraviolet (UV) absorbance measurements of the tyrosine chromophore. The versatility of NMR for the quantitative analysis of peptides that do not contain an appropriate UV chromophore make it well-suited for the determination of peptide concentration in aggregation studies or for the preparation of solutions for high-throughput screening of biological activity.