title: Evaluation of online carbon isotope dilution mass spectrometry for the purity assessment of synthetic peptide standards.
abstract: We present a novel method for the purity assessment of peptide standards which is applicable to any water soluble peptide. The method is based on the online (13)C isotope dilution approach in which the peptide is separated from its related impurities by liquid chromatography (LC) and the eluent is mixed post-column with a continuous flow of (13)C-enriched sodium bicarbonate. An online oxidation step using sodium persulfate in acidic media at 99°C provides quantitative oxidation to (12)CO2 and (13)CO2 respectively which is extracted to a gaseous phase with the help of a gas permeable membrane. The measurement of the isotope ratio 44/45 in the mass spectrometer allows the construction of the mass flow chromatogram. As the only species that is finally measured in the mass spectrometer is CO2, the peptide content in the standard can be quantified, on the base of its carbon content, using a generic primary standard such as potassium hydrogen phthalate. The approach was validated by the analysis of a reference material (NIST 8327), and applied to the quantification of two commercial synthetic peptide standards. In that case, the results obtained were compared with those obtained using alternative methods, such as amino acid analysis and ICP-MS. The results obtained proved the value of the method for the fast, accurate and precise mass purity assignment of synthetic peptide standards.
authors.affiliation: Department of Physical and Analytical Chemistry, University of Oviedo, Julián Clavería 8, 33006 Oviedo, Spain.
authors.forename: Sergio
authors.initials: S
authors.lastname: Cueto Díaz
authors.affiliation: Department of Physical and Analytical Chemistry, University of Oviedo, Julián Clavería 8, 33006 Oviedo, Spain. Electronic address: ruizjorge@uniovi.es.
authors.forename: Jorge
authors.initials: J
authors.lastname: Ruiz Encinar
authors.affiliation: Department of Physical and Analytical Chemistry, University of Oviedo, Julián Clavería 8, 33006 Oviedo, Spain. Electronic address: jiga@uniovi.es.
authors.forename: J Ignacio
authors.initials: JI
authors.lastname: García Alonso
mesh_terms: D055316:Cystatin C / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry* / Q000592:standards; D012015:Reference Standards; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin / Q000737:chemistry
chemical_list: D055316:Cystatin C; D010455:Peptides; D014357:Trypsin
keywords: Isotope dilution mass spectrometry; Peptide standards; Quantitative proteomics; Traceability