An LC-MS Approach to Quantitative Measurement of Ammonia Isotopologues

Abstract

Ammonia is a fundamental aspect of metabolism spanning all of phylogeny. Metabolomics, including metabolic tracing studies, are an integral part of elucidating the role of ammonia in these systems. However, current methods for measurement of ammonia are spectrophotometric, and cannot distinguish isotopologues of ammonia, significantly limiting metabolic tracing studies. Here, we describe a novel LC-MS-based method that quantitatively assesses both 14N-and 15N-isotopologues of ammonia in polar metabolite extracts. This assay (1) quantitatively measures the concentration of ammonia in polar metabolite isolates used for metabolomic studies, and (2) accurately determines the percent isotope abundance of 15N-ammonia in a cell lysate for 15N-isotope tracing studies. We apply this assay to quantitatively measure glutamine-derived ammonia in lung cancer cell lines with differential expression of glutaminase.

Transaminase Inhibition by 2-Hydroxyglutarate Impairs Glutamate Biosynthesis and Redox Homeostasis in Glioma

“…Tracing 15 N-(amide)-glutamine into ammonia was performed as previously described (Spinelli et al, 2017). Briefly, HOG stable cell lines were given 15 N-(amide)-glutamine for 8 hours before metabolites were extracted with 80:20 methanol:water and assessed for 15 NH 3 .…”

“…Briefly, HOG stable cell lines were given 15 N-(amide)-glutamine for 8 hours before metabolites were extracted with 80:20 methanol:water and assessed for 15 NH 3 . Ammonia was derivatized into indophenol with the Berthelot reaction, as previously described (Spinelli et al, 2017), and measured on an Agilent mass spectrometer coupled to reverse phase ion-pairing chromatography. Percent 15 N-isotopologue enrichment was measured by dividing the ion counts of 15 N-indophenol by the sum of 14 N and 15 N isotopologues of indophenol.…”

Metabolic recycling of ammonia via glutamate dehydrogenase supports breast cancer biomass

“…Next, we measured the amount of 15 NH 3 generated after 8 hour treatment with 15 N-(amide)-glutamine using an LC-MS method for detection of 15 NH 3 from 15 N-(amide)-glutamine (23). In MCF7 and T47D cells, only 3.5% of the total ammonia pool derived from glutaminolysis (fig.…”

Serum Metabolic Profiling Identifies a Biomarker Panel for Improvement of Prostate Cancer Diagnosis

“…Moreover, the spectrometry technology has been improved a lot recently. More details of molecular structure tend to be observed by using this method and speci c protocol [8,9,10]. For example, the use of nano ow liquid chromatography-mass spectrometry (nanoLC-MS) makes it possible to do the pro ling of the glycans and the glycolipids which can be used in the clinic studies, including glycan-based biomarker discovery and therapeutics.…”