Rawi Ramautar (Presenter)
Leiden University
Bio: Dr. Rawi Ramautar is an assistant professor at the Leiden Academic Center for Drug Research (LACDR) of the Leiden University. He received his M.Sc. degrees in Pharmacochemistry and Analytical Sciences from the Vrije Universiteit Amsterdam in 2004. He received his Ph.D. in Analytical Chemistry from the Utrecht University in 2010, under the guidance of Prof. Dr. G.J. de Jong and Prof. Dr. G.W. Somsen. He developed capillary electrophoresis-mass spectrometry (CE-MS) methods using non-covalently coated capillaries for metabolic profiling studies. Dr. Rawi Ramautar continued his studies as a Postdoctoral Fellow in the group of Prof. André Deelder and Dr. Oleg Mayboroda at the Leiden University Medical Center where he developed CE-MS approaches for the comprehensive analysis of metabolites in clinical samples. In 2012, Dr. Rawi Ramautar accepted a position at the LACDR of Leiden University whe
Authorship: Rawi Ramautar
Leiden Academic Center for Drug Research, Leiden University
| Short Abstract A critical need for clinical metabolomics is a microscale analytical platform for the selective analysis of highly polar metabolites in small-volume samples. Capillary electrophoresis-mass spectrometry (CE-MS) is an attractive approach for this purpose, as compounds are separated according to their charge-to-size ratio and nanoliter injection volumes are employed. A diverse range of polar endogenous metabolites including separation of important isomers could be efficiently analyzed at the (sub)nanomolar level using a novel sheathless nanospray interface for coupling CE to MS. The utility is shown for metabolic profiling of ultra-small samples like cerebrospinal fluid from mice. Overall, the proposed microscale analytical platform opens a way for a deeper understanding of biological functions in sample-limited cases. |
Long Abstract
In contemporary clinical metabolomics, a multitude of high-end analytical technologies are used for the global profiling of endogenous metabolites in biological samples. However, a reliable analytical methodology for the selective and efficient analysis of highly polar and charged metabolites is still lacking. A critical need for clinical metabolomics is also the introduction of microscale analytical methods allowing the analysis of those samples for which the amount is severely limited. Capillary electrophoresis (CE) hyphenated to mass spectrometry (MS) can be regarded an attractive microscale analytical platform for this purpose, as in CE nanoliter injection volumes are employed from sub-microliter sample amounts. Since compounds are separated on the basis of their charge-to-size ratio, CE is fundamentally different from chromatographic-based separation techniques, providing a complementary view on the composition of a biological sample. Moreover, CE provides very high peak efficiencies and is highly suited for the analysis of low-abundance polar and charged metabolites in ultra-small samples.
Here, the performance of CE coupled to electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) via a novel sheathless nanospray interface is demonstrated for the global profiling of ionogenic metabolites in small-volume biological samples. The intrinsically low-flow characteristics of CE are effectively utilized in this set-up, enabling ESI-TOF-MS to reach its optimal performance, i.e. an improved sensitivity and reduced ion suppression. As a consequence, the enhanced sensitivity of the sheathless CE-TOF-MS method allows comprehensive analysis of polar and charged metabolites in biological samples. The applicability is shown for ultra-small biological samples like cerebrospinal fluid (CSF) from mice using direct sample injection, which is of pivotal importance for non-targeted metabolic profiling, and for neuronal cells generated from human-induced pluripotent stem cells.
Sheathless interfacing of a CE instrument and a high-end TOF-MS instrument was achieved through a capillary of which the outlet section was etched with hydrofluoric acid, providing an outer terminus porous to the electric transport of small ions. The separation capillary with this porous tip was placed in a stainless steel ESI needle filled with static conductive liquid composed of 10% acetic acid. ESI was performed in positive (−1.5 kV) and negative ionization mode (+1.5 kV) using 10% acetic acid (pH 2.2) as background-electrolyte. MS data were recorded in the 65-1000 m/z region. Representative metabolite mixtures, mouse CSF, glioblastoma and neuronal cell cultures were used for method evaluation.
The use of sheathless CE-TOF-MS under low-flow separation conditions with an injection volume of only ca. 9 nanoliter resulted in low nanomolar detection limits for a broad array of metabolite classes, such as sugar phosphates, organic acids, nucleotides, nucleosides, amino acids and small peptides. Structural isomers of phosphorylated sugars as well as isobaric metabolites could be selectively analyzed by the sheathless CE-TOF-MS method without using any derivatization. An efficient separation of these compounds is key in order to allow selective detection by MS. A unique feature of the proposed approach is that both highly polar anionic and cationic metabolites could be efficiently analyzed by only switching the MS detection and CE separation voltage polarity.
The potential of sheathless CE-TOF-MS for in-depth metabolic profiling of volume-limited samples was investigated for mouse CSF, of which only a few microliters can be obtained under proper experimental conditions, and for samples containing only a limited number of cells (~10000 cells/mL). It is shown that highly information-rich metabolic profiles could be obtained for these limited amounts of biological samples. For example, approximately 300 molecular features (S/N>10) were detected in mouse CSF, whereas about 100 molecular features (S/N>5) were found in a glioblastoma cell line extract with a cell density of 20 cells/nL by the sheathless CE-TOF-MS methodology.
Compared to other analytical techniques, such as reversed-phase LC-MS and GC-MS, a significant amount of complementary metabolomics information is provided by the sheathless CE-MS method, especially concerning the analysis of the highly polar phosphorylated sugars and nucleotides. Concerning body fluids such as urine and CSF, metabolic profiling by sheathless CE-TOF-MS can be performed with minimal sample pretreatment (only 1:1 dilution with water), thereby fully retaining sample integrity which is of pivotal importance for the global and unbiased profiling of polar and charged metabolites.
Although the utility of the proposed analytical methodology in clinical metabolomics has only been shown for a limited set of samples so far, the studies have provided important and useful advances in this rapidly evolving field. Overall, it is anticipated that the sheathless CE-TOF-MS platform will allow important clinical metabolomics studies that have so far been lacking and it will open ways for a deeper understanding of biological functions in sample-limited cases.
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