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Development of a novel approach for quantification of amino acids in complex biological fluids
Author(s) -
Johnson Jennifer M.,
Strobel Fred,
Jones Dean P
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a708-c
Subject(s) - amino acid , chemistry , chromatography , yeast , mass spectrometry , isoleucine , leucine , biochemistry
Recent advances in mass spectrometry have allowed for rapid metabolic analysis of complex biological fluids such as plasma; however, reliable quantification of hundreds to thousands of metabolites remains a challenge. The present study was to determine whether yeast could be used to create a complex biological isotopic standard. Commercial baker's yeast was grown in broth containing normal glucose and ammonium sulfate or isotopic 13 C‐glucose and 15 N‐ammonium sulfate through 8 doublings to achieve 98% isotope incorporation. Cells were extracted with −40° methanol and metabolites were separated with HPLC coupled to a high resolution Fourier transform mass spectrometry (FTMS). To test the feasibility of this approach, analysis focused on 15 common amino acids. Peaks corresponding to the theoretical accurate mass of all 15 isotopic amino acids were observed. An amino acid standard mixture (Sigma), spiked into the isotopic mixture, was used to generate standard curves and quantify the amino acids. Concentrations were found to be in a biologically normal range, with high concentrations of glutamate (103μM) and leucine/isoleucine (95μM) and low concentrations of tyrosine (1.4μM) and tryptophan (0.14μM). These values correspond to published free amino acid concentrations in yeast. These results show that stable isotopic yeast extract can be used as a standard for complex biological fluids.