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Capillary Electrophoresis-Mass Spectrometry for Micro-Metabolomics
Wei Zhang1 1
and Rawi Ramautar1 *
Biomedical Microscale Analytics, Division of Systems Biomedicine and Pharmacology, Leiden Academic Centre for Drug Research, Leiden University, the Netherlands *Corresponding author: Dr Rawi Ramautar, Email:
r.ramautar@lacdr.leidenuniv.nl
To address biomedical questions intrinsically dealing with limited sample amounts with a metabolomics approach, the design of novel or improved analytical techniques is needed. In this paper, we highlight the possibilities of capillary electrophoresis-mass spectrometry (CE-MS) as a microscale analytical platform for metabolic profiling of small amounts of biological material. A few examples from my group are discussed in order to show the utility of CE-MS for this purpose. Finally, we reflect on a number of analytical challenges that still need to be addressed in order to make CE-MS a viable approach for material-limited metabolomics.
Introduction
Metabolomics has become an important tool in biomedical and clinical research and is particularly promising in biomarker discovery. Currently, reversed-phase LC-MS, GC-MS and nuclear magnetic resonance spectroscopy (NMR) are used as the main analytical tools for metabolomics studies. These analytical techniques require sample material which often ranges in the amount from 10 to 50 µL for LC-MS and GC-MS (in this case needed for both sample preparation and injection), and up to 500 µL for NMR. Consequently, the need for these minimal sample amounts often prevents their use for biomedical and clinical problems inherently dealing with very low amounts of material [1-3]. For example, metabolic profiling of cerebrospinal fluid (CSF) from transgenic mouse models, spheroids/microtissues, liquid biopsies and samples from microfluidic 3D cell culture models, is seriously hindered by the standard analytical technologies.
Until now, relatively little effort has been made to downscale the analytical technique and /or workflow for metabolomics studies, as the application has been often focused on addressing biomedical and clinical questions associated with human urine and plasma (or serum) samples. In order to enable micro-metabolomics, i.e. the metabolomics study of biological
and biomedical questions intrinsically dealing with small amounts of sample material, new analytical technology with a dramatically improved sensitivity is needed. The approach of micro-metabolomics was first coined by Moco et al. in the field of plant metabolomics [4], however, here we refer to this approach in the context of biomedical and clinical questions. It is our conviction that the successful development of a microscale analytical platform for material-restricted biological samples will constitute a real breakthrough in the field of metabolomics as it will open the way for a deeper understanding of biological processes in sample-limited cases.
CE-MS is a preferred analytical method for extracting (more) chemical information using less material
Capillary electrophoresis-mass spectrometry (CE-MS) can be considered an attractive microscale analytical technique for addressing biological questions inherently dealing with low amounts of material [5-10]. An example that cannot be properly studied with the current standard analytical tools, and which is of particular interest to our group, concerns the disentanglement of the behaviour of a single cell within a group or
population of mammalian cells, and as such to obtain a better understanding on the role of cell heterogeneity in tumour biology.
In CE, nanolitre injection volumes can be used from just a few microliters of sample or less in the injection vial. Therefore, CE-MS can be regarded as a highly suited approach for the analysis of especially polar and charged metabolites in tiny sample amounts, as demonstrated by our group for example for mouse CSF [11]. This body fluid can only be obtained in a few microliters under the right experimental conditions. By employing a 1:1 dilution of CSF with water, and as a result completely retaining sample integrity, more than 300 compounds could be detected by CE-MS. For the injection only 45 nL of the sample was consumed from a vial containing not more than 2 µL of a 1:1 diluted CSF. Therefore, the CE-MS approach allows multiple analyses on a single highly valuable mouse CSF sample, enabling repeatability studies and, even more interestingly, to analyse the same sample at different separation conditions in order to enhance resolution and therefore metabolic coverage. The ability to perform multiple analyses on a single scarcely available biological sample is not possible with the conventional analytical techniques employed in metabolomics.
In CE, analytes are separated on the basis of differences in their intrinsic electrophoretic mobilities which is,
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