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Quantitative 1H nuclear magnetic resonance (qNMR) of aromatic amino acids for protein quantification
- Hydrolysis of protein samples into amino acids facilitates the use of NMR spectroscopy for protein and peptide quantification. Different conditions have been tested for quantifying aromatic amino acids and proteins. The pH-dependent signal shifts in the aromatic region of amino acid samples were examined. A pH of 12 was found to minimize signal overlap of the four aromatic amino acids. Several aromatic compounds, such as terephthalic acid, sulfoisophthalic acid, and benzene tricarboxylic acid, were applied as internal standards. The quantification of amino acids from an amino acid standard was performed. Using the first two suggested internal standards, recovery was ~97% for histidine, phenylalanine, and tyrosine at a concentration of approximately 1 mM in solution. Acidic hydrolysis of a certified reference material (CRM) of bovine serum albumin (BSA) and subsequent quantification of Phe and Tyr yielded recoveries of 98% ± 2% and 88% ± 4%, respectively, at a protein concentration ofHydrolysis of protein samples into amino acids facilitates the use of NMR spectroscopy for protein and peptide quantification. Different conditions have been tested for quantifying aromatic amino acids and proteins. The pH-dependent signal shifts in the aromatic region of amino acid samples were examined. A pH of 12 was found to minimize signal overlap of the four aromatic amino acids. Several aromatic compounds, such as terephthalic acid, sulfoisophthalic acid, and benzene tricarboxylic acid, were applied as internal standards. The quantification of amino acids from an amino acid standard was performed. Using the first two suggested internal standards, recovery was ~97% for histidine, phenylalanine, and tyrosine at a concentration of approximately 1 mM in solution. Acidic hydrolysis of a certified reference material (CRM) of bovine serum albumin (BSA) and subsequent quantification of Phe and Tyr yielded recoveries of 98% ± 2% and 88% ± 4%, respectively, at a protein concentration of 16 g/L or 250 µM.…
Autor*innen: | Teodor Tchipilov, Klas MeyerORCiD, Michael G. WellerORCiD |
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Dokumenttyp: | Zeitschriftenartikel |
Veröffentlichungsform: | Verlagsliteratur |
Sprache: | Englisch |
Titel des übergeordneten Werkes (Englisch): | Methods and protocols |
Jahr der Erstveröffentlichung: | 2023 |
Organisationseinheit der BAM: | 1 Analytische Chemie; Referenzmaterialien |
1 Analytische Chemie; Referenzmaterialien / 1.4 Prozessanalytik | |
1 Analytische Chemie; Referenzmaterialien / 1.5 Proteinanalytik | |
Veröffentlichende Institution: | Bundesanstalt für Materialforschung und -prüfung (BAM) |
Verlag: | MDPI |
Verlagsort: | Basel, Schweiz |
Jahrgang/Band: | 6 |
Ausgabe/Heft: | 1 |
Aufsatznummer: | 11 |
Erste Seite: | 1 |
Letzte Seite: | 13 |
DDC-Klassifikation: | Naturwissenschaften und Mathematik / Chemie / Analytische Chemie |
Freie Schlagwörter: | AAA; AAAA; Amino acid analysis; Aromatic amino acid analysis; Benzene-1,3,5-tricarboxylic acid; Bovine serum albumin (BSA); Calibration; Compound-independent calibration; Histidine; Hydrochloric acid; Internal standard; Metrology; NIST; Phenylalanine; Phenylketonuria; Protein hydrolysis; Quantitative protein analysis; Reference materials; Terephthalic acid; Traceability; Tryptophan; Tyrosine |
Themenfelder/Aktivitätsfelder der BAM: | Chemie und Prozesstechnik |
Chemie und Prozesstechnik / Chemische Charakterisierung und Spurenanalytik | |
DOI: | 10.3390/mps6010011 |
URN: | urn:nbn:de:kobv:b43-570943 |
Zugehöriger Identifikator: | https://nbn-resolving.org/urn:nbn:de:kobv:b43-564520 |
Verfügbarkeit des Dokuments: | Datei für die Öffentlichkeit verfügbar ("Open Access") |
Lizenz (Deutsch): | Creative Commons - CC BY - Namensnennung 4.0 International |
Datum der Freischaltung: | 09.03.2023 |
Referierte Publikation: | Ja |
Datum der Eintragung als referierte Publikation: | 09.03.2023 |
Schriftenreihen ohne Nummerierung: | Wissenschaftliche Artikel der BAM |