|Improving Untargeted Metabolomics with Ion Chromatography-Mass Spectrometry
|Hyphenated Techniques for Comprehensive Analysis
|Mr John Walsby-Tickle
|Dr Joan Gannon
Ms Elisabete Pires
Dr David Hauton
Prof James McCullagh
|University of Oxford
Abstract Information :
Metabolomics is a rapidly growing area of mass spectrometry (MS) which maps molecular changes in cells, tissues and bio-fluids. Liquid chromatography (LC) coupled with mass spectrometry (LC-MS) is frequently used to identify molecular phenotypes associated with diseases such as cancer and the effect of pharmacological intervention on metabolic pathways. The principal LC separation methods currently used are reversed-phase (RP) and hydrophilic interaction chromatography (HILIC). HILIC has been shown to perform well for polar metabolites in both targeted and untargeted analysis due to its solvent compatibility with MS and relatively fast run times. However, it requires careful column equilibration and pH balance to maintain a delicate aqueous layer on the stationary phase, leading to poor retention time reproducibility across large numbers of samples.
To date ion chromatography (IC) coupled directly to MS (IC-MS) is relatively rare in metabolomics but has a number of characteristics which makes it an attractive chromatographic approach for coverage of highly polar and ionic metabolites. We have been developing this technique for targeted and untargeted applications and have demonstrated exceptional retention time stability and lower limits of detection when compared to HILIC. This performance is due to on-line eluent generation from water and a concentrated ion solution which leads to highly reproducible chromatographic conditions and low chemical noise. IC-MS also requires minimal sample preparation as cell lysates and bio-fluids can be run directly after filtration, minimising metabolite degradation and intra-sample variability.
Here we demonstrate a number of advantages in using IC as part of multiplatform LC-MS for untargeted metabolomics applied to the profiling of metabolites from glioblastoma cells expressing isocitrate dehydrogenase 1 (IDH1) mutations. IDH1 is a change of function mutation which disrupts the normal citric acid cycle conversion of isocitrate to 2-oxoglutarate (2-OG) and catalyses the production of 2-hydroxyglutarate (2-HG) from both species. IC is an ideal method for the separation of tricarboxylic acid cycle metabolites due to their high polarity and similar functionality which present a challenge for analysis using standard chromatographic techniques.
We analysed cell extracts from multiple glioblastoma cell lines with and without the IDH1 mutation in different media conditions. Three LC-MS approaches were used to ensure maximal coverage of the metabolome: IC, RP and derivatised RP. This ensured that consecutive pathways could be followed and the cumulative impact of pathway modification measured. Our aim was to determine whether metabolic changes occurred as a result of the presence of the IDH1 mutation and if any such changes were also affected by glucose concentration. Here we show the power of IC for untargeted metabolomics and the effect of glucose concentration and IDH1 mutation on the metabolome, especially on glycolysis and pentose phosphate pathways.