Elsevier

Talanta

Volume 152, 15 May 2016, Pages 119-126
Talanta

Characterization of oncogene-induced metabolic alterations in hepatic cells by using ultrahigh performance liquid chromatography-tandem mass spectrometry

https://doi.org/10.1016/j.talanta.2016.01.056Get rights and content

Highlights

  • An UHPLC-MS/MS-based metabolomics was established to elucidate altered metabolism.

  • Metabolic perturbations in EIF5A2-transfected LO2 cells were figured out.

  • Levels of lactate and ATP in EIF5A2-transfected LO2 cells were dramatically altered.

  • Patterns of amino acid imbalance in EIF5A2-transfected LO2 cells were observed.

Abstract

Elucidation of altered metabolic pathways by using metabolomics may open new avenues for basic research on disease mechanisms and facilitate the development of novel therapeutic strategies. Here, we report the development of ultrahigh performance liquid chromatography-tandem mass spectrometry-based metabolomics platform with capability of measuring both cationic and anionic intermediates in cellular metabolism. The platform was established based on the hydrophobic ion-pairing interaction chromatography coupled with tandem mass spectrometry in multiple reaction monitoring (MRM) mode. The MRM transitions were created and optimized via energy-resolved collision-induced dissociation experiments, serving as an essential reference point for the quantification and identification. For chromatographic separation, application of hydrophobic ion-pairing interaction led to dramatic enhancement on retention of water-soluble metabolites and provision of good peak shapes. Two volatile ion-pairing reagents, namely heptafluorobutyric acid and tributylamine, were used with dedicated C18 columns as complementary separation systems coupled with the MRM analysis, allowing measurement of the metabolites of interest at nanomolar levels. The developed platform was successfully applied to investigate the altered metabolism in hepatic cells with over-expression of an oncogene, thus can provide important information on the rewired metabolism.

Introduction

Reprogramming cellular metabolism is an emerging hallmark of cancer [1], which is characterized by high glycolysis even under aerobic conditions, known as the Warburg effect [2]. Growing number of abnormal metabolic patterns in different types of cancer including breast [3], lung [4], leukemias [5] and pancreas [6] are reported, which are mechanistically linked to the malfunctions of metabolic enzymes and the alterations of oncogenes or tumor suppressor genes [7]. Fundamentally, the altered metabolism may offer promising opportunities for the development of novel therapeutics to target cancer cell metabolism [8]. Thus, more explorations to unravel the specific metabolic features in various conditions are expected for further advancement by means of multidisciplinary approach.

Metabolites play a key role in cancer metabolism, either to serve as nutrients for energy production or precursors for macromolecular biosynthesis [2]. Accordingly, the systematic survey of metabolites, termed as metabolomics, is considered to provide a direct functional readout of the biochemical activity or (patho)physiological status of an organism [9]. Among the analytical technologies employed for measuring the metabolites, mass spectrometry coupled to different separation techniques, such as capillary electrophoresis [10], gas chromatography [11], [12] and high performance liquid chromatography [13], [14], have become the major tools to analyze a wide range of metabolites of interest. Considering the remarkable differences in abundance and chemical diversity, as well as the coexistence of isomeric and isobaric metabolites, tandem mass spectrometry coupled to liquid chromatography has been desirable principally due to the high separation efficiency with adjustable selectivity through changes in the mobile and stationary phase in separation systems [15], [16], [17], [18] and high sensitivity as well as selectivity provided by tandem mass spectrometry [19], [20]. It is a remarkable fact that tandem mass spectrometry coupled with ultrahigh performance liquid chromatography (UHPLC-MS/MS) serves as a key tool enabling fast and sensitive analysis of small molecules in complex biological systems [16], [19]. Development of UHPLC-MS/MS-based metabolomics platform capable of analyzing a broad range of metabolites in diverse biological processes, including cancer studies, is continuously increasing and expected.

Within living cells, a core set of metabolic events, called the central carbon metabolism, including glycolysis, the tricarboxylic acid (TCA) cycle (also known as the citric acid cycle or the Krebs cycle), pentose phosphate pathway (PPP), lays foundation for the generation of energy and reducing power and the production of intermediates and biosynthetic precursors that are essential for cell growth, proliferation and survival [1], [2], [21]. Therefore, in this work, mass spectrometry-based metabolomics method was developed and demonstrated for hepatic cell metabolism studies. By choosing immortal hepatic cell line LO2 as an in vitro experimental model, we focus on targeted metabolomics for analyzing glycolysis and its interconnected pathways, to elucidate oncogene-induced metabolic reprogramming. The method used heptafluorobutyric acid (HFBA) and tributylamine (TBA) as volatile ion-pairing reagents in two reversed-phase liquid chromatography systems with complementary separation selectivity and coupled to electrospray ionization-triple quadrupole tandem mass spectrometry operated in positive and negative ion modes, respectively. The developed method has been successfully applied to measure central carbon metabolites in LO2 cell line transfected with the oncogene-eukaryotic translation initiation factor 5A2 (EIF5A2).

Section snippets

Chemicals and reagents

Methanol and acetonitrile were HPLC grade and obtained from Tedia (Fairfield, OH, USA). Ultrapure water was purified by a Milli-Q Academic Water Purification System from Millipore (Millipore, Bedford, MA, USA). TBA (Purity: 99%) was purchased from Acros Organics (New Jersey, USA). HFBA (Purity ≥99.5%), and the highest purity commercially available metabolite standards were obtained from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification (Supporting information, Table A1).

Results and discussion

In this work, to achieve the goal of establishing a mass spectrometry-based targeted metabolomics platform for cancer studies, we concentrated our efforts on developing triple-quadrupole mass spectrometry coupled to UHPLC for measurements of the central carbon metabolism and amino acid metabolism in cultured human cells.

Conclusion

An UHPLC-MS/MS-based targeted metabolomics platform has been established for the elucidation of altered cancer metabolism with the complementary use of volatile ion-pairing reagents (HFBA for cationic species and TBA for anionic species) as mobile phase modifiers in UHPLC coupled to MRM analysis. As a proof-of-concept, the potential of applying the MRM-based targeted metabolomics to elucidate the oncogene EIF5A2 induced metabolic alterations in human hepatic liver cells was demonstrated, and

Acknowledgment

This work was supported by Hong Kong Research Grant Council Collaborative Research Fund (HKBU5/CRF/10) and the National Natural Science Foundation of China (NSFC-21377106).

References (27)

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