Can Edman degradation be used for quantification? Isotope-dilution liquid chromatography-electrospray ionization tandem mass spectrometry and the long-term stability of 20 phenylthiohydantoin-amino acids Amino Acid Analysis

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Abstract

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 [13C6]-PTH-AAs were prepared by use of a one-pot reaction involving a mixture of [13C6]-Edman reagent and 20 AAs. Good linearity was observed for standard curves for the PTH-AAs, using the corresponding [13C6]-PTH-AAs as internal standards (1-100 pmol per injection, r 2 = 0.989-1.000). Serum albumin (human), pepsin (porcine stomach mucosa), α-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. [Figure not available: see fulltext.]

Original languageEnglish
Pages (from-to)8001-8010
Number of pages10
JournalAnalytical and Bioanalytical Chemistry
Volume405
Issue number25
DOIs
Publication statusPublished - 2013 Oct

Keywords

  • Absolute quantification
  • Edman degradation
  • Phenylthiohydantoin-amino acids

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry

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