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

被引:4
|
作者
Tchipilov, Teodor [1 ]
Meyer, Klas [2 ]
Weller, Michael G. [1 ]
机构
[1] BAM Fed Inst Mat Res & Testing, Div Prot Anal 1.5, Richard Willstatter-Str 11, D-12489 Berlin, Germany
[2] BAM Fed Inst Mat Res & Testing, Div Proc Analyt Technol 1.4, Richard Willstatter-Str 11, D-12489 Berlin, Germany
关键词
amino acid analysis; AAA; protein hydrolysis; metrology; traceability; reference materials; internal standards; calibration; MASS-SPECTROMETRY; DERIVATIZATION; SULFUR; PURITY; COULD;
D O I
10.3390/mps6010011
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 similar to 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 mu M.
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页数:13
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