A positive/negative ion-switching, targeted mass spectrometry-based metabolomics platform for bodily fluids, cells, and fresh and fixed tissue

Abstract

The revival of interest in cancer cell metabolism in recent years has prompted the need for quantitative analytical platforms for studying metabolites from in vivo sources. We implemented a quantitative polar metabolomics profiling platform using selected reaction monitoring with a 5500 QTRAP hybrid triple quadrupole mass spectrometer that covers all major metabolic pathways. The platform uses hydrophilic interaction liquid chromatography with positive/negative ion switching to analyze 258 metabolites (289 Q1/Q3 transitions) from a single 15-min liquid chromatography-mass spectrometry acquisition with a 3-ms dwell time and a 1.55-s duty cycle time. Previous platforms use more than one experiment to profile this number of metabolites from different ionization modes. The platform is compatible with polar metabolites from any biological source, including fresh tissues, cancer cells, bodily fluids and formalin-fixed paraffin-embedded tumor tissue. Relative quantification can be achieved without using internal standards, and integrated peak areas based on total ion current can be used for statistical analyses and pathway analyses across biological sample conditions. The procedure takes ∼12 h from metabolite extraction to peak integration for a data set containing 15 total samples (∼6 h for a single sample).

Figure 1

Metabolomics platform schematic. Overview of the targeted LC-MS/MS experiment for polar metabolite profiling via SRM, using positive/negative switching from a single 15-min HILIC column run while targeting more than 250 compounds.

Figure 2

Reproducibility and heat maps from cancer cell metabolomics. (a) Reproducibility of cancer cell extracts by polar metabolomics profiling platform. Typical R² values across biological replicates in a data set from 293T embryonic kidney and H929 multiple myeloma cells are shown. (b) Unsupervised hierarchical clustering heat map of metabolites from both H929 and 293T cancer cells stimulated with the growth factors EGF and insulin across different time points (0, 15, 30 min). (c) PCA clustering of polar metabolites from the same 293T and H929 human cancer cell extracts with EGF and insulin stimulation, showing different clustering groups according to incubation time of growth factor stimulation. (d) Fold changes of individual glycolytic intermediates upon acute serum stimulation in 293T and H929 cells. Western blots of key signaling nodes such as phosphorylated AKT (pAKT) correlate with glucose uptake in cancers.

Figure 3

In vivo metabolomics profiling. (a) The PCA clustering from extracted polar metabolites from cerebrospinal fluid (CSF) from 20 patients, 10 of which present with gliomas. Different disease stages are highlighted. (b) Hierarchical clustergram from polar metabolites that were extracted from formalin-fixed paraffin-embedded (FFPE) tissue cores from normal (Norm) lung and kidney tissue and from acute myeloid leukemia (AML) from kidney and lymphangioleiomyomatosis (LAM) lung disease. The data show that distinct clusters can be obtained from fixed tissue samples.