Mass Spectrometric Method for Analyzing Metabolites in Yeast with Single Cell Sensitivity

Abstract

Almost all currently employed analytical "-omics" methods provide data that are averaged over an entire cell population, but even genetically identical cells exposed to the same environmental conditions can show strong variations in molecular content and even in phenotypes. [1] Such heterogeneity is involved in many cellular and also disease-related processes, such as antibiotic resistance, [2] competence for DNA uptake, [3] and in viral life-cycle decisions. [4] The increasing number of such findings suggests that cellular heterogeneity is underestimated, which in turn calls for development of novel single-cell-based analytical methods.Today, several techniques for chemical analysis of single cells exist, most of which focus on detection of peptides and proteins. In addition to the high sensitivity that is generally required for single cell analysis, the detection of intracellular metabolites (typically small molecules with MW < 1000 Da), poses additional challenges. In contrast to the protein and transcription levels, the metabolic level presents 1) turnover rates on the order of seconds, thus demanding fast cell processing, 2) a large chemical diversity, and 3) small molecular weights, which renders fluorescent tagging difficult without impacting the biological function of metabolites. Only very few studies have so far demonstrated the feasibility of small-molecule analysis in single cells. Capillary electrophoresis coupled with laser-induced fluorescence, a method named metabolic cytometry, [5] has been successfully used to monitor the oligosaccharide cascade of biodegradation products generated in single yeast cells, [6] in HT29 colon cancer cells, [5] and has recently been employed to characterize the metabolism of sphingolipids in single pituitary tumor cells. [7] Fluorescence resonance energy transfer (FRET) sensors have also been used to detect small molecules, such as glutamate, [8] in single cells. A variety of other techniques, for example, laser-induced native fluorescence [9] and capillary electrophoresis coupled with voltammetric detection [10] and UV adsorption [11] have been utilized to detect neurotransmitters and amino acids in single cells. What most of the currently existing approaches to small molecule analysis in single cells have in common, however, is that they are tailored to analyze a specific metabolite of interest, not allowing a broad measurement of many different metabolites at the same time, which is the goal of the emerging field of metabolomics.In the field of classical (that is, population-averaging) metabolomics, mass spectrometry (MS) is increasingly used because, owing to its versatility, it can cope with the large chemical diversity of the metabolome. Although MS has been employed for single-cell analysis, [12] in particular for peptides and proteins, and in two studies detected very highly concentrated metabolites (histamine and serotonin in single mast cells [13] ), MS has so far been severely limited for the detection of endogenous metabolites in single cells because o...

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Metabolic Differences in Microbial Cell Populations Revealed by Nanophotonic Ionization

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