PubMed · 10664579
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S J Freeland, R D Knight, L F Landweber. 2000. Measuring adaptation within the genetic code.. https://doi.org/10.1016/s0968-0004(99)01531-5
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Amino acid (AA) profiles from body fluids such as blood and urine are clinical indicators for diagnosing metabolic and hepatic diseases. Current quantitative methods, such as liquid chromatography-mass spectrometry (LC-MS) with isotopically labelled internal standards (ISs), are costly and technically demanding. This study proposes a cost-efficient alternative using structural isomers as ISs in a direct liquid infusion (DLI) tandem mass spectrometry (MS/MS) approach. The method leverages chimeric spectra and fragment intensity ratios to quantify AAs, demonstrating high linearity and precision even with a 3D ion trap mass analyser. This approach offers a viable strategy for AA quantification in preventive medicine, particularly for screening metabolic diseases such as phenylketonuria, diabetes, and liver dysfunction.
An on-line high-pressure liquid chromatography (HPLC) system capable of measuring amino acids and carbohydrates was used to study metabolism in mammalian cell culture systems. The HPLC method utilized anion-exchange chromatography followed by integrated pulsed amperometric detection. The method is capable of measuring 19 amino acids plus glucose with a complete method time of 65 min. In actual cell cultures, the method was shown to be useful for monitoring 17 amino acids plus glucose. The two amino acids that were not accurately monitored were arginine and lysine, possibly due to their elution near the void volume of the column. The HPLC system was used to study variability in metabolism across different cell culture processes, as well as the effect of glucose and glutamine limitation on a single cell culture process. Chemometric analysis was used to draw statistically meaningful conclusions from the highly correlated, multivariate data set that resulted from these experiments. Using chemometrics, variation between processes was linked to differences in uptake rates of seven amino acids. Similarly, lactate concentration, cell density, and aspartate uptake rate were linked to glucose and glutamine limitation. The effect of nutrient limitation on glutamate, alanine, and ammonium was also considered.
Recombinant protein overexpression and the classical stringent response have been shown to induce the same proteases. Since the stringent response was the result of an intracellular amino acid shortage, it was hypothesized that the overexpression of the recombinant protein also caused an intracellular amino acid shortage. A structured non-segregated kinetic mathematical model of recombinant Escherichia coli was developed to predict intracellular amino acid shortages during recombinant protein overexpression, and thus the induction of the stringent response. Two model recombinant proteins were examined, chloramphenicol acetyl-transferase (CAT) and an 'average protein'. The model predicted an aromatic amino acid shortage during CAT overexpression, as predicted based on the CAT's amino acid content. The model also predicted a shortage of the intracellular alanine family amino acid pool during CAT overexpression. This was unexpected due to the relatively low content of alanine family amino acids in CAT compared to the average E. coli protein. The model predicted alanine, glutarate, and aspartate family amino acid shortages during recombinant 'average protein' overexpression. Additionally, the model predicted a decrease in the ribosome pool at induction for both recombinant proteins, which agrees with published experimental results. Thus, the structured kinetic model was able to predict amino acid shortages, that could potentially cause a stringent response and elevated protease activity.