Sheathless capillary electrophoresis-mass spectrometry as a new approach for analyzing the polar metabolome
Metabolomicshas emerged as an important discipline to study molecular and cellular processes in living cells and organisms with the ultimate aim to obtain an answer to a given biological/clinical question.
- Rawi Ramautar
NWO Veni grant nr.: 722.013.008
Today, modern analytical techniques, such as nuclear magnetic resonance spectroscopy and mass spectrometry coupled to liquid chromatography or gas chromatography, are generally used for the global and reproducible profiling of endogenous metabolites in biological samples. Despite important innovations in analytical technology over the past years the current analytical toolbox still encounters difficulties during the analysis of highly polar and charged metabolites, i.e. the polar metabolome.
In order to address this issue, the group of Dr. Rawi Ramautar recently developed a new approach for the highly sensitive, efficient and selective analysis of highly polar metabolites (e.g. sugar phosphates, small organic acids and nucleotides) in complex sample matrices. The approach is based on capillary electrophoresis (CE) coupled online to electrospray ionization (ESI) MS. In CE, analytes are separated on the basis of their charge-to-size ratio making it an attractive tool for the profiling of highly polar and charged metabolites. For the coupling to ESI-MS, Dr. Ramautar has used a new sheathless interface design, which was invented by Dr. Mehdi Moini, in order to fully exploit the intrinsically low-flow property of CE as a significantly improved sensitivity and reduced ion suppression can be obtained under these conditions.
The performance of the analytical tool was evaluated for anionic metabolic profiling studies. Representative metabolite mixtures and biological samples were used for the evaluation of various analytical parameters. For test compounds, relative standard deviations for migration times and peak areas were below 2% and 11%, respectively, and plate numbers ranged from 60000 to 400000 demonstrating a high separation efficiency. Critical metabolites with low or no retention on reversed-phase LC could be efficiently separated and selectively analysed by the sheathless CE-MS method. An injection volume of only circa 20 nL resulted in nanomolar detection limits for a wide range of metabolites.
The methodology was applied to metabolic profiling of glioblastoma cell line extracts. Sheathless CE-MS method could also be used for the global profiling of cationic metabolites (i.e. amino acids, amines, nucleosides and small peptides) by only switching the MS detection and separation voltage polarity.
The proposed approach is also suited for small-volume metabolomics studies. Therefore, Dr. Ramautar expects that further development in this research field will have a major impact in metabolomics as the new analytical approach will open a new direction, i.e. towards a deeper understanding of biological functions in sample-restricted cases.