Developing liquid chromatography-mass spectrometry strategies for investigating energy metabolism with application to isocitrate dehydrogenase mutations in cancer

Isocitrate dehydrogenases (IDHs) act in the tricarboxylic acid (TCA) cycle to catalyse the conversion of isocitrate to 2-oxoglutarate (2-OG) with concomitant production of NADH and/or NADPH. In humans, mutations causing IDH1 and IDH2 substitutions have been found during DNA sequencing of human glioblastomas. Some cancerassociated IDH mutations promote the reduction of 2-OG to give D-2-hydroxyglutarate (D-2-HG) a chiral, polar dicarboxylic acid.

Investigations on the underlying links between 2-HG and cancer require sensitive methods for monitoring IDH1 and IDH2 activities. The reported LC/MS methods for the analysis of TCA cycle metabolites are limited in term of their accuracy and ability to identity isomers. Methods for the direct measurement of levels of TCA cycle and associated metabolites using mass spectrometry based metabolomics are desired. However, for the reported methods, the limits of detection are often prohibitive; they are normally suited for the investigation of known metabolites rather than for the discovery of new compounds. With some exceptions, the existing methods have not been extensively validated, e.g. with respect to limits of detection and quantification as well as precision. The main aim of the research presented in this thesis was to develop methods for TCA cycle metabolite analysis in cells and to apply these methods to investigations on variant IDH enzymes.

A range of LC-MS approaches were investigated including: (1) C18 reversed phase chromatography of non-derivatised TCA cycle metabolites, (2) ion paring chromatography, and (3) mixed mode chromatography with either MS, or isotope ratio mass spectrometry detection, gas chromatography of TBDMS derivatised TCA cycle metabolites. Analysis of the elution patterns for these separation techniques enabled estimation of the retention parameters of TCA cycle metabolites and investigations on their metabolism. The most sensitive approach developed employed mixed mode chromatography coupled to isotope ratio mass spectrometry, which was optimised for the analysis of TCA cycle metabolites. This was shown to have a limit of detection two orders of magnitude lower (4μM) than more conventional mass spectrometry techniques. Using 13C-[4C]-Aspartate labelling in cell culture, a quantification protocol was developed which employed a non-labelled internal standard and selectively labelled cell culture. The method was shown to be suitable for both very accurate quantification at low concentration levels and metabolic studies.

The analytical methods developed for TCA cycle metabolites analysis were successfully applied to the analysis of 2-OG and D-2-HG metabolism. The stereochemistry of 2-HG in the cell pellets as well as of the citrate/isocitrate isomers was investigated. IDH1R132H was shown to catalyse reduction of 2-OG resulting in D-2-HG. TCA cycle analysis was used in order to investigate 2-OG and 2-HG metabolism related to IDH1R132H. Using the method developed for the analysis of non-derivatised TCA cycle intermediates, the screening of potential substrates of IDH1R132H was carried out. The isotope ratio mass spectrometry protocol was applied to the study of IDH1R132H in cell culture; levels of D-2-HG were quantified.

The analytical methods described complement the established metabolomics techniques. The methods developed enable the investigation into the regio- and stereo- chemistry of TCA cycle and associated metabolites and are powerful tools for investigating cancer cell metabolism.

Advisor: McCullagh, J
Schofield, C