3 Capillary Electrophoresis-Mass Spectrometry for Micro-Metabolomics Wei Zhang1 and Rawi Ramautar1* 1Biomedical Microscale Analytics, Division of Systems Biomedicine and Pharmacology, Leiden Academic Centre for Drug Research, Leiden University, the Netherlands *Corresponding author: Dr Rawi Ramautar, Email: [email protected] 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 and biomedical questions intrinsically population of mammalian cells, and as such dealing with small amounts of sample to obtain a better understanding on the role Metabolomics has become an important material, new analytical technology with a of cell heterogeneity in tumour biology. tool in biomedical and clinical research dramatically improved sensitivity is needed. and is particularly promising in biomarker In CE, nanolitre injection volumes can be The approach of micro-metabolomics was discovery. Currently, reversed-phase LC-MS, used from just a few microliters of sample first coined by Moco et al. in the field of GC-MS and nuclear magnetic resonance or less in the injection vial. Therefore, plant metabolomics [4], however, here we spectroscopy (NMR) are used as the main CE-MS can be regarded as a highly suited refer to this approach in the context of analytical tools for metabolomics studies. approach for the analysis of especially polar biomedical and clinical questions. It is our These analytical techniques require sample and charged metabolites in tiny sample conviction that the successful development material which often ranges in the amount amounts, as demonstrated by our group for of a microscale analytical platform for from 10 to 50 μL for LC-MS and GC-MS example for mouse CSF [11]. This body fluid material-restricted biological samples will (in this case needed for both sample can only be obtained in a few microliters constitute a real breakthrough in the field preparation and injection), and up to 500 under the right experimental conditions. By of metabolomics as it will open the way μL for NMR. Consequently, the need for employing a 1:1 dilution of CSF with water, for a deeper understanding of biological these minimal sample amounts often and as a result completely retaining sample processes in sample-limited cases. prevents their use for biomedical and clinical integrity, more than 300 compounds could problems inherently dealing with very low be detected by CE-MS. For the injection amounts of material [1-3]. For example, CE-MS is a preferred only 45 nL of the sample was consumed from a vial containing not more than 2 μL metabolic profiling of cerebrospinal fluid analytical method for (CSF) from transgenic mouse models, of a 1:1 diluted CSF. Therefore, the CE-MS spheroids/microtissues, liquid biopsies extracting (more) approach allows multiple analyses on a and samples from microfluidic 3D cell chemical information single highly valuable mouse CSF sample, culture models, is seriously hindered by the using less material enabling repeatability studies and, even more interestingly, to analyse the same standard analytical technologies. Capillary electrophoresis-mass spectrometry sample at different separation conditions in Until now, relatively little effort has been (CE-MS) can be considered an attractive order to enhance resolution and therefore made to downscale the analytical technique microscale analytical technique for metabolic coverage. The ability to perform and /or workflow for metabolomics addressing biological questions inherently multiple analyses on a single scarcely studies, as the application has been often dealing with low amounts of material [5-10]. available biological sample is not possible focused on addressing biomedical and An example that cannot be properly studied with the conventional analytical techniques clinical questions associated with human with the current standard analytical tools, employed in metabolomics. urine and plasma (or serum) samples. In and which is of particular interest to our order to enable micro-metabolomics, group, concerns the disentanglement of the In CE, analytes are separated on the i.e. the metabolomics study of biological behaviour of a single cell within a group or basis of differences in their intrinsic electrophoretic mobilities which is, 4 February / March 2019 Figure 1. Extracted ion electropherograms for selected endogenous metabolites detected by sheathless CE-MS in extracts from HepG2 cells using starting amounts from 10,000 to 500 cells (Figure 1A and 1C). The detector response for the selected metabolites as a function of the starting amount of HepG2 cells used for analysis is shown in Figure 1B and 1D. Experimental conditions: MS detection in positive ion mode; CE was coupled to MS via a sheathless porous tip emitter; BGE, 10% acetic acid (pH 2.2); Separation voltage: 30 kV; sample injection: 6.0 psi for 60 s. except for the low sample and solvent studies were often focused on the analysis HepG2 cells as a model system [23]. requirement of CE, fundamentally different of a relatively large single non-mammalian The aim was to profile a wide range of from chromatographic-based analytical cell (diameter in the range of 100 to 1000 endogenous metabolites in just a few techniques. Consequently, CE-MS provides μm with a cellular sample content ranging cells, as the latter will enable to study a complementary view on the composition from circa 10 to 1000 nL). The content of a the effect of cell heterogeneity, which of endogenous metabolites present in a single mammalian cell is estimated to be really matters in various key fundamental given biological sample [12]. Compared to around 1 pL (diameter ~10 μm) [13], and biological questions. The sheathless porous chromatographic-based separation methods that of a single HepG2 cell is estimated tip interface was developed by Moini a the separation efficiency of CE is very high to be around 3 pL (diameter ~12 μm). decade ago by removing the polyimide due to the flat flow profile of the electro- Therefore, the profiling of (endogenous) coating of a fused-silica capillary outlet osmotic flow. Moreover, there is no mass metabolites in a single mammalian cell is via etching of the capillary wall with 49% transfer between phases and therefore only clearly a vast analytical challenge. Over the solution of hydrofluoric acid to a thickness longitudinal diffusion contributes to band past few years, various research groups have of about 5 μm [24]. The electrical connection broadening under proper experimental done pioneering work in the development to the capillary outlet is achieved in this conditions. The intrinsically high separation of analytical techniques for acquiring configuration by inserting the etched efficiency of CE is very advantageous for metabolic profiles from a single cell [14-19]. conductor into an ESI needle which is filled the high resolution separation of structurally For an overview of these technological with separation buffer. The sheathless similar metabolites in complex samples. developments, we refer to the following porous tip design is especially useful for recent reviews [20-22]. interfacing narrow (<30 μm inner diameter) Until now, various research groups have capillaries and for low flow-rate (<100 nL/ developed CE-MS approaches for metabolic In my group, we have recently assessed the min) nano-ESI-MS analyses. profiling of limited sample amounts, utility of CE-MS employing a sheathless and also for single cell analysis [3, 10]. porous tip interface for metabolic profiling By the integration of an in-capillary Concerning the latter, the metabolomics of low numbers of mammalian cells using preconcentration technique, in this 5 case transient isotachophoresis, in the sample vials, pipette tips, etc.), notably Conflict of Interest sheathless CE-MS approach low nanomolar with sample volumes far below 1 μL, The authors have no other relevant detection limits could be easily obtained may result in significant analyte losses. affiliations or financial involvement with any for a wide range of basic metabolites (e.g., A more critical factor is the fraction of organisation or entity with a financial interest amino acids, amines, small peptides). It the sample that needs to be (effectively) in or financial conflict with the subject matter is important to stress that low nanomolar injected into the CE-MS system. In this or materials discussed in the manuscript detection limits could be obtained by only context, miniaturisation and optimisation apart from those disclosed. using an injection volume of circa 40 nL of sample preparation will be crucial for (total capillary volume is ~600 nL). In cases material-limited metabolomics studies. where sample volume is not an issue LC-MS For CE-MS-based metabolomics studies, References will be a preferred analytical technique, fractionation procedures based on liquid- however, it is important to highlight that liquid extraction for the selective isolation 1. Fessenden, M., Nature