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

Abstract

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, 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 C 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, R > 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 C 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.

Merging metabolomics and lipidomics into one analytical run

“…41 For untargeted data processing of metabolites, Thermo Scientific Compound Discoverer software was used as described in a previous publication. 42 For untargeted data analysis of lipids, LipidSearch from Thermo Scientific was used. Details on the identification are given in the ESI.…”

“…A recent IC-HRMS study showed that simultaneous absolute quantification of 45 metabolites and non-targeted analysis was successfully performed in cancer cells upon integration of yeast derived 13 C labeled standards. 42 The number of annotated metabolites was not significantly influenced by the presence of an internal standard. Following the analogous strategy in this work, the metabolome extraction procedures comprised the addition of the yeast derived 13 C internal standard with the aim of performing both simultaneously, absolute quantification of >50 primary metabolites and non-targeted fingerprinting.…”

“…Histidine eluted as an extremely broad peak of more than eight minutes, while spermine and spermidine could not be detected. With respect to anion chromatography, 42 the metabolite panel could be extended to amino acids and other positively charged metabolites. In this work, HILIC was adapted to short gradient times and tested for a standard panel of 133 metabolites (see Table S1 †).…”

“…Overall, the limits of detection were comparable to those of a previously published ion-exchange chromatography method. 42 Moreover, excellent recoveries were assessed for 1 µM quality control (QC) standards (containing the 13 C labeled yeast extract) representing the trueness bias of quantification by external and internal standards. A significant fraction of the studied metabolites could be determined using both ionization modes, while organic acids, sugars and sugar phosphates required negative ionization and a minor fraction of intrinsically positively charged metabolites (nicotinamide, carnitine, and propionylcarnitine) could only be measured in the positive mode.…”

The Power of LC-MS Based Multiomics: Exploring Adipogenic Differentiation of Human Mesenchymal Stem/Stromal Cells

“…For targeted analysis, we employed 13 C isotopically labeled metabolite and lipid standards produced in our laboratory for standardization [15,28,29] to follow fat cell differentitation of MSCs. Absolute compound-specific quantification of 12 lipids and 54 metabolites was performed using external calibration with internal standardization by in-house produced 13 C-labeled standards and protein normalization.…”