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 requirement of CE, fundamentally different from chromatographic-based analytical techniques. Consequently, CE-MS provides a complementary view on the composition of endogenous metabolites present in a given biological sample [12]. Compared to chromatographic-based separation methods the separation efficiency of CE is very high due to the flat flow profile of the electro- osmotic flow. Moreover, there is no mass transfer between phases and therefore only longitudinal diffusion contributes to band broadening under proper experimental conditions. The intrinsically high separation efficiency of CE is very advantageous for the high resolution separation of structurally similar metabolites in complex samples.
Until now, various research groups have developed CE-MS approaches for metabolic profiling of limited sample amounts, and also for single cell analysis [3, 10]. Concerning the latter, the metabolomics
studies were often focused on the analysis of a relatively large single non-mammalian cell (diameter in the range of 100 to 1000 µm with a cellular sample content ranging from circa 10 to 1000 nL). The content of a single mammalian cell is estimated to be around 1 pL (diameter ~10 µm) [13], and that of a single HepG2 cell is estimated to be around 3 pL (diameter ~12 µm). Therefore, the profiling of (endogenous) metabolites in a single mammalian cell is clearly a vast analytical challenge. Over the past few years, various research groups have done pioneering work in the development of analytical techniques for acquiring metabolic profiles from a single cell [14-19]. For an overview of these technological developments, we refer to the following recent reviews [20-22].
In my group, we have recently assessed the utility of CE-MS employing a sheathless porous tip interface for metabolic profiling of low numbers of mammalian cells using
HepG2 cells as a model system [23]. The aim was to profile a wide range of endogenous metabolites in just a few cells, as the latter will enable to study the effect of cell heterogeneity, which really matters in various key fundamental biological questions. The sheathless porous tip interface was developed by Moini a decade ago by removing the polyimide coating of a fused-silica capillary outlet via etching of the capillary wall with 49% solution of hydrofluoric acid to a thickness of about 5 µm [24]. The electrical connection to the capillary outlet is achieved in this configuration by inserting the etched conductor into an ESI needle which is filled with separation buffer. The sheathless porous tip design is especially useful for interfacing narrow (<30 µm inner diameter) capillaries and for low flow-rate (<100 nL/ min) nano-ESI-MS analyses.
By the integration of an in-capillary preconcentration technique, in this
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