Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Quantitative 1H Nuclear Magnetic Resonance (qNMR) of Aromatic Amino Acids for Protein Quantification

Version 1 : Received: 28 November 2022 / Approved: 30 November 2022 / Online: 30 November 2022 (09:17:12 CET)

A peer-reviewed article of this Preprint also exists.

Tchipilov, T.; Meyer, K.; Weller, M.G. Quantitative 1H Nuclear Magnetic Resonance (QNMR) of Aromatic Amino Acids for Protein Quantification. Methods and Protocols 2023, 6, 11, doi:10.3390/mps6010011. Tchipilov, T.; Meyer, K.; Weller, M.G. Quantitative 1H Nuclear Magnetic Resonance (QNMR) of Aromatic Amino Acids for Protein Quantification. Methods and Protocols 2023, 6, 11, doi:10.3390/mps6010011.

Abstract

qNMR is a valuable technique for metrological studies due to the uniformity of its signal response for all chemical species of an isotope of interest, which enables compound-independent calibration. However, protein quantification remained challenging as large molecules produce wide, low-intensity signals that reduce the already low sensitivity. Combining qNMR with the hydrolysis of protein samples into amino acids circumvents many of these issues and facilitates the use of NMR spectroscopy for absolute protein and peptide quantification.In this work, different conditions have been tested for quantifying aromatic amino acids and proteins. First, we examined the pH-based signal shifts in the aromatic region. The preferable pH depends on the selection of the amino acids for quantification and which internal standard substance should be used to avoid peak overlap. Several aromatic compounds, such as terephthalic acid, sulfoisophthalic acid, and benzene tricarboxylic acid, have been applied as internal standards. The quantification of amino acids from an amino acid standard, as well as from a certified reference material (bovine serum albumin), 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.

Keywords

amino acid analysis; AAA; protein hydrolysis; metrology; traceability; reference materials; internal standards; calibration

Subject

Biology and Life Sciences, Biology and Biotechnology

Comments (0)

We encourage comments and feedback from a broad range of readers. See criteria for comments and our Diversity statement.

Leave a public comment
Send a private comment to the author(s)
* All users must log in before leaving a comment
Views 0
Downloads 0
Comments 0
Metrics 0


×
Alerts
Notify me about updates to this article or when a peer-reviewed version is published.
We use cookies on our website to ensure you get the best experience.
Read more about our cookies here.