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Protein Quantification by Derivatization-Free High-Performance Liquid Chromatography of Aromatic Amino Acids

  • Amino acid analysis is considered to be the gold standard for quantitative peptide and protein analysis. Here, we would like to propose a simple HPLC/UV method based on a reversed-phase separation of the aromatic amino acids tyrosine (Tyr), phenylalanine (Phe), and optionally tryptophan (Trp) without any derivatization. The hydrolysis of the proteins and peptides was performed by an accelerated microwave technique, which needs only 30 minutes. Two internal standard compounds, homotyrosine (HTyr) and 4-fluorophenylalanine (FPhe) were used for calibration. The limit of detection (LOD) was estimated to be 0.05 µM (~10 µg/L) for tyrosine and phenylalanine at 215 nm. The LOD for a protein determination was calculated to be below 16 mg/L (~300 ng BSA absolute). Aromatic amino acid analysis (AAAA) offers excellent accuracy and a precision of about 5% relative standard deviation, including the hydrolysis step. The method was validated with certified reference materials (CRM) of amino acids andAmino acid analysis is considered to be the gold standard for quantitative peptide and protein analysis. Here, we would like to propose a simple HPLC/UV method based on a reversed-phase separation of the aromatic amino acids tyrosine (Tyr), phenylalanine (Phe), and optionally tryptophan (Trp) without any derivatization. The hydrolysis of the proteins and peptides was performed by an accelerated microwave technique, which needs only 30 minutes. Two internal standard compounds, homotyrosine (HTyr) and 4-fluorophenylalanine (FPhe) were used for calibration. The limit of detection (LOD) was estimated to be 0.05 µM (~10 µg/L) for tyrosine and phenylalanine at 215 nm. The LOD for a protein determination was calculated to be below 16 mg/L (~300 ng BSA absolute). Aromatic amino acid analysis (AAAA) offers excellent accuracy and a precision of about 5% relative standard deviation, including the hydrolysis step. The method was validated with certified reference materials (CRM) of amino acids and of a pure protein (bovine serum albumin, BSA). AAAA can be used for the quantification of aromatic amino acids, isolated peptides or proteins, complex peptide or protein samples, such as serum or milk powder, and peptides or proteins immobilized on solid supports.zeige mehrzeige weniger
Metadaten
Autor*innen:Almut Hesse, Michael G. WellerORCiD
Dokumenttyp:Zeitschriftenartikel
Veröffentlichungsform:Verlagsliteratur
Sprache:Englisch
Titel des übergeordneten Werkes (Englisch):Journal of Amino Acids
Jahr der Erstveröffentlichung:2016
Veröffentlichende Institution:Bundesanstalt für Materialforschung und -prüfung (BAM)
Verlag:Hindawi
Verlagsort:Cairo, London, New York
Jahrgang/Band:2016
Erste Seite:Article 7374316, 1
Letzte Seite:8
DDC-Klassifikation:Naturwissenschaften und Mathematik / Chemie / Analytische Chemie
Freie Schlagwörter:Amino acid analysis; Certified reference materials; Hydrolysis; Metrology; Microwave; Peptides; Phenylalanine; Protein analysis; Proteomics; Tryptophan; Tyrosine
DOI:10.1155/2016/7374316
URN:urn:nbn:de:kobv:b43-370215
ISSN:2090-0112
ISSN:2090-0104
Verfügbarkeit des Dokuments:Datei für die Öffentlichkeit verfügbar ("Open Access")
Lizenz (Deutsch):License LogoCreative Commons - Namensnennung
Datum der Freischaltung:01.08.2016
Referierte Publikation:Ja
Datum der Eintragung als referierte Publikation:26.06.2017
Schriftenreihen ohne Nummerierung:Wissenschaftliche Artikel der BAM
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