Measurement of the 15N/14N ratio of phenylalanine in fermentation matrix by isotope ratio mass spectrometry
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To determine the origin of 15N-labeled phenylalanine in microbial metabolic flux analysis using 15N as a tracer, a method for measuring phenylalanine δ15N using HPLC coupled with elemental analysis-isotope ratio mass spectrometry (EA-IRMS) was developed.
The original source of the 15N-labeled phenylalanine was determined using this new method that consists of three steps: optimization of the HPLC conditions, evaluation of the isotope fractionation effects, and evaluation of the effect of pre-processing on the phenylalanine nitrogen stable isotope. In addition, the use of a 15N-labeled inorganic nitrogen source, rather than 15N-labeled amino acids, was explored using this method.
The method described here can also be applied to the analysis of metabolic flux.
KeywordsElemental analysis-isotope ratio mass spectrometry (EA-IRMS) High-performance liquid chromatography Metabolic flux analysis Phenylalanine δ15N
This study was supported by the International S&T Cooperation Program of China (2015DFA31720), the National Nature Foundation of China (31601580) and the National Science and Technology Project of the Ministry of Science and Technology in the 13th Five-Year Plan Period (2016YFF0203903).
Supplementary Fig. 1—Preparative separation of microbial hydrolysis mixture.
Supplementary Fig. 2—Measured δ15N value of phenylalanine added with phenylalanine standard material.
Supplementary Fig. 3—15N phenylalanine enrichment is in linear relationship with 15N-labeled NH4Cl concentration.
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Conflict of interest
The authors declare that they have no conflict of interest.
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