Isotope-dilution liquid chromatography-electrospray ionization tandem mass spectrometry and the long-term stability of 20 phenylthiohydantoin-amino acids

Edman degradation is a well-known method for obtaining amino acid (AA) sequences from a peptide by means of sequential reactions that release the N-terminal AAs from the peptide as a phenylthiohydantoin (PTH) derivative. Because of unexpected loss during the reaction and handling, there are few reports of use of this reaction for quantification. This manuscript describes the development of isotope-dilution liquid chromatography-electrospray ionization tandem mass spectrometry for 20 PTH-AA derivatives, and long-term stability testing of PTH-AAs to ensure quantitative quality in the reaction. The 20 corresponding [(13)C(6)]-PTH-AAs were prepared by use of a one-pot reaction involving a mixture of [(13)C(6)]-Edman reagent and 20 AAs. Good linearity was observed for standard curves for the PTH-AAs, using the corresponding [(13)C(6)]-PTH-AAs as internal standards (1-100 pmol per injection, r(2)=0.989-1.000). Serum albumin (human), pepsin (porcine stomach mucosa), alpha -casein (bovine milk), ribonuclease A (bovine), lysozyme (chicken egg white), and insulin (bovine) subjected to Edman degradation were examined as model proteins and peptides for N-terminal AA analysis. The results of the impurity test were satisfactory. Yield from the entire reaction with human serum albumin was estimated to be at least 75 %, indicating great potential for absolute quantification of proteins without protein standards.