Anion-Exchange Chromatography Coupled to High-Resolution
Mass Spectrometry: A Powerful Tool for Merging Targeted and Non-targeted Metabolomics
Posted on 2017-06-05 - 00:00
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.
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Schwaiger, Michaela; Rampler, Evelyn; Hermann, Gerrit; Miklos, Walter; Berger, Walter; Koellensperger, Gunda (2017). Anion-Exchange Chromatography Coupled to High-Resolution
Mass Spectrometry: A Powerful Tool for Merging Targeted and Non-targeted Metabolomics. ACS Publications. Collection. https://doi.org/10.1021/acs.analchem.7b01624
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AUTHORS (6)
MS
Michaela Schwaiger
ER
Evelyn Rampler
GH
Gerrit Hermann
WM
Walter Miklos
WB
Walter Berger
GK
Gunda Koellensperger