title: 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.
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 [(13)C6]-PTH-AAs were prepared by use of a one-pot reaction involving a mixture of [(13)C6]-Edman reagent and 20 AAs. Good linearity was observed for standard curves for the PTH-AAs, using the corresponding [(13)C6]-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.
authors.affiliation: Department of Bio-analytical Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aramaki-aoba, Aoba-ku, Sendai, 980-8578, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Satoh
authors.forename: Takaaki
authors.initials: T
authors.lastname: Goto
authors.forename: Seon Hwa
authors.initials: SH
authors.lastname: Lee
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Oe
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D002247:Carbon Isotopes / Q000737:chemistry; D002417:Cattle; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007201:Indicator Dilution Techniques; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds / Q000737:chemistry*; D010669:Phenylthiohydantoin / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D013552:Swine; D053719:Tandem Mass Spectrometry
chemical_list: C043517:2-(4-isothiocyanatophenoxy)-1,3,2-dioxaphosphinene 2-oxide; D000596:Amino Acids; D002247:Carbon Isotopes; D009943:Organophosphorus Compounds; D011506:Proteins; D010669:Phenylthiohydantoin