5
case transient isotachophoresis, in the sheathless CE-MS approach low nanomolar detection limits could be easily obtained for a wide range of basic metabolites (e.g., amino acids, amines, small peptides). It is important to stress that low nanomolar detection limits could be obtained by only using an injection volume of circa 40 nL (total capillary volume is ~600 nL). In cases where sample volume is not an issue LC-MS will be a preferred analytical technique, however, it is important to highlight that reversed-phase LC-MS is not a suitable tool for the analysis of highly polar and charged metabolites.
Given the nanomolar concentration sensitivity, the sheathless CE-MS method could be effectively used for acquiring metabolic profiles in HepG2 cells starting from 10,000 down to 500 cells, which corresponds to the injection content of 5 HepG2 cells to less than one cell. These results suggest that the method has the sensitivity for performing single cell mammalian metabolomics studies. Though, the sheathless CE-MS method has been primarily used as a screening method so far, we have also compared the detector response (using peak area as read-out) for a few selected endogenous metabolites as a function of the starting amount of HepG2 cells. As shown in Figure 1, this analysis revealed a linear detector response (R2
=0.9986 and 0.9948 for
S-adenosyl methionine and glutamic acid, respectively) when going from 10,000 to 500 HepG2 cells, indicating the potential of the sheathless CE-MS method for quantitative metabolomics studies of limited sample amounts. The performance of the approach in terms of repeatability for profiling metabolites in small amounts of HepG2 cells was assessed by the consecutive analyses of a single extract from 10,000 HepG2 cells. The relative standard deviation (RSD) values for migration times and peak areas of selected endogenous metabolites were below 3% and 10%, respectively, which are acceptable values for studies employed under ultra-low flow-rate separation conditions in conjunction with nano-ESI-MS conditions.
Analytical challenges associated with material- limited metabolomics
Metabolomics studies dealing with low amounts of biological sample have to critically consider pre-analytical steps as adsorption effects (for example, to
sample vials, pipette tips, etc.), notably with sample volumes far below 1 µL, may result in significant analyte losses. A more critical factor is the fraction of the sample that needs to be (effectively) injected into the CE-MS system. In this context, miniaturisation and optimisation of sample preparation will be crucial for material-limited metabolomics studies. For CE-MS-based metabolomics studies, fractionation procedures based on liquid- liquid extraction for the selective isolation of polar and charged metabolites (which obviously will be in the polar phase), from proteins and non-polar compounds in small amounts of biological samples can be considered. The polar phase/fraction can be further processed using enrichment tools as electroextraction or depleted zone isotachophoresis [25-27]. For example, in electroextraction, analytes can be extracted from a donor phase, i.e. the sample, into an acceptor phase by using an electric field. Alternatively, polar and charged metabolites can be selectively enriched from a biological sample via an organic layer into a hanging aqueous droplet prior to further analysis. With depletion zone isotachophoresis, charged compounds can be preconcentrated and separated in a microfluidic channel according to their electrophoretic mobilities, adjacent to a depleted zone created by a nanochannel. Afterwards, the charged compounds can be released in fractions according to their electrophoretic mobility for further analysis. In the proposed sample preparation steps, the fractionated polar and charged compounds can be further separated by the sheathless CE-MS method.
Overall, the combination of an effective, tailor-made sample preparation method with sheathless CE-MS will enable us to acquire metabolic profiles from a low number of mammalian cells, ultimately a single mammalian cell, in a robust way. We anticipate that such a fully optimised microscale analytical workflow will be of high value to nanodosing studies and single cell biopsies, especially in the context of cancer research.
Acknowledgements
Wei Zhang would like to acknowledge the China Scholarship Council (CSC, No. 201507060011). Rawi Ramautar would like to acknowledge the financial support of the Vidi grant scheme of the Netherlands Organization for Scientific Research (NWO Vidi 723.016.003).
Conflict of Interest
The authors have no other relevant affiliations or financial involvement with any organisation or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
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