4 years ago

Anion-Exchange Chromatography Coupled to High-Resolution Mass Spectrometry: A Powerful Tool for Merging Targeted and Non-targeted Metabolomics

Anion-Exchange Chromatography Coupled to High-Resolution Mass Spectrometry: A Powerful Tool for Merging Targeted and Non-targeted Metabolomics
Evelyn Rampler, Michaela Schwaiger, Walter Berger, Walter Miklos, Gerrit Hermann, Gunda Koellensperger
In this work, simultaneous targeted metabolic profiling by isotope dilution and non-targeted fingerprinting is proposed for cancer cell studies. The novel streamlined metabolomics workflow was established using anion-exchange chromatography (IC) coupled to high-resolution mass spectrometry (MS). The separation time of strong anion-exchange (2 mm column, flow rate 380 μL min–1, injection volume 5 μL) could be decreased to 25 min for a target list comprising organic acids, sugars, sugar phosphates, and nucleotides. Internal standardization by fully 13C labeled Pichia pastoris extracts enabled absolute quantification of the primary metabolites in adherent cancer cell models. Limits of detection (LODs) in the low nanomolar range and excellent intermediate precisions of the isotopologue ratios (on average <5%, N = 5, over 40 h) were observed. As a result of internal standardization, linear dynamic ranges over 4 orders of magnitude (5 nM–50 μM, R2 > 0.99) were obtained. Experiments on drug-sensitive versus resistant SW480 cancer cells showed the feasibility of merging analytical tasks into one analytical run. Comparing fingerprinting with and without internal standard proved that the presence of the 13C labeled yeast extract required for absolute quantification was not detrimental to non-targeted data evaluation. Several interesting metabolites were discovered by accurate mass and comparing MS2 spectra (acquired in ddMS2 mode) with spectral libraries. Significant differences revealed distinct metabolic phenotypes of drug-sensitive and resistant SW480 cells.

Publisher URL: http://dx.doi.org/10.1021/acs.analchem.7b01624

DOI: 10.1021/acs.analchem.7b01624

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