Single-cell heterogeneity in metabolism, drug resistance and disease type poses the need for analytical techniques for single-cell analysis. As the metabolome provides the closest view of the... Show moreSingle-cell heterogeneity in metabolism, drug resistance and disease type poses the need for analytical techniques for single-cell analysis. As the metabolome provides the closest view of the status quo in the cell, studying the metabolome at single-cell resolution may unravel said heterogeneity. A challenge in single-cell metabolome analysis is that metabolites cannot be amplified, so one needs to deal with picolitre volumes and a wide range of analyte concentrations. Due to high sensitivity and resolution, MS is preferred in single-cell metabolomics. Large numbers of cells need to be analysed for proper statistics; this requires high-throughput analysis, and hence automation of the analytical workflow. Significant advances in (micro)sampling methods, CE and ion mobility spectrometry have been made, some of which have been applied in high-throughput analyses. Microfluidics has enabled an automation of cell picking and metabolite extraction; image recognition has enabled automated cell identification. Many techniques have been used for data analysis, varying from conventional techniques to novel combinations of advanced chemometric approaches. Steps have been set in making data more findable, accessible, interoperable and reusable, but significant opportunities for improvement remain. Herein, advances in single-cell analysis workflows and data analysis are discussed, and recommendations are made based on the experimental goal. Show less
Mever, M. van; Fabjanowicz, M.; Mamani-Huanca, M.; Lopez-Gonzalvez, A.; Plotka-Wasylka, J.; Ramautar, R. 2022
The composition of wine is determined by a complex interaction between environmental factors, genetic factors (i.e., grape varieties), and winemaking practices (including technology and storage).... Show moreThe composition of wine is determined by a complex interaction between environmental factors, genetic factors (i.e., grape varieties), and winemaking practices (including technology and storage). Metabolomics using NMR spectroscopy, GC-MS, and/or LC-MS has shown to be a useful approach for assessing the origin, authenticity, and quality of various wines. Nonetheless, the use of additional analytical techniques with complementary separation mechanisms may aid in the deeper understanding of wine's metabolic processes. In this study, we demonstrate that CE-MS is a very suitable approach for the efficient profiling of polar ionogenic metabolites in wines. Without using any sample preparation or derivatization, wine was analyzed using a 10-min CE-MS workflow with interday RSD values for 31 polar and charged metabolites below 3.8% and 23% for migration times and peak areas, respectively. The utility of this workflow for the global profiling of polar ionogenic metabolites in wine was evaluated by analyzing different cool-climate Polish wine samples. Show less
Zhang, W.; Guled, F.; Hankemeier, T.; Ramautar, R. 2020
Negative ion mode nano-ESI-MS is often considered for the analysis of acidic compounds, including nucleotides. However, under high aqueous separation conditions, corona discharge is frequently... Show moreNegative ion mode nano-ESI-MS is often considered for the analysis of acidic compounds, including nucleotides. However, under high aqueous separation conditions, corona discharge is frequently observed at emitter tips, which may result in low ion abundances and reduced nano-ESI needle emitter lifetimes. In this work, we introduce a sheathless CE-MS method for the highly efficient and sensitive analysis of nucleotides employing ESI in positive ion mode, thereby fully circumventing corona discharge. By using a background electrolyte of 16 mM ammonium acetate (pH 9.7) a mixture of 12 nucleotides, composed of mono-, di-, and tri-phosphates, could be efficiently analyzed with plate numbers per meter above 220 000 and with LODs in the range from 0.06 to 1.3 nM, corresponding to 0.4 to 8.6 attomole, when using an injection volume of about 6.5 nL only. The utility of the method was demonstrated for the profiling of nucleotides in low numbers of mammalian cells using HepG2 cells as a model system. Endogenous nucleotides could be efficiently analyzed in extracts from 50 000 down to 500 HepG2 cells only. Moreover, apart from nucleotides, also some nicotinamide-adenine dinucleotides and amino acids could be analyzed under these conditions, thereby clearly illustrating the utility of this approach for metabolic profiling of low amounts of biological material. Show less
The efficient profiling of highly polar and charged metabolites in biological samples remains a huge analytical challenge in metabolomics. Over the last decade, new analytical techniques have been... Show moreThe efficient profiling of highly polar and charged metabolites in biological samples remains a huge analytical challenge in metabolomics. Over the last decade, new analytical techniques have been developed for the selective and sensitive analysis of polar ionogenic compounds in various matrices. Still, the analysis of such compounds, notably for acidic ionogenic metabolites, remains a challenging endeavor, even more when the available sample size becomes an issue for the total analytical workflow. In this paper, we give an overview of the possibilities of capillary electrophoresis-mass spectrometry (CE-MS) for anionic metabolic profiling by focusing on main methodological developments. Attention is paid to the development of improved separation conditions and new interfacing designs in CE-MS for anionic metabolic profiling. A complete overview of all CE-MS-based methods developed for this purpose is provided in table format (Table 1) which includes information on sample type, separation conditions, mass analyzer and limits of detection (LODs). Selected applications are discussed to show the utility of CE-MS for anionic metabolic profiling, especially for small-volume biological samples. On the basis of the examination of the reported literature in this specific field, we conclude that there is still room for the design of a highly sensitive and reliable CE-MS method for anionic metabolic profiling. A rigorous validation and the availability of standard operating procedures would be highly favorable in order to make CE-MS an alternative, viable analytical technique for metabolomics. Show less
Zhang, W.; Segers, K.R.H.; Mangelings, D.; Eeckhaut, A. van; Hankemeier, T.; Heyden, Y. van der; Ramautar, R. 2019
The actual utility of capillary electrophoresis-mass spectrometry (CE-MS) for biomarker discovery using metabolomics still needs to be assessed. Therefore, a simulated comparative metabolic... Show moreThe actual utility of capillary electrophoresis-mass spectrometry (CE-MS) for biomarker discovery using metabolomics still needs to be assessed. Therefore, a simulated comparative metabolic profiling study for biomarker discovery by CE-MS was performed, using pooled human plasma samples with spiked biomarkers. Two studies have been carried out in this work. Focus of study I was on comparing two sets of plasma samples, in which one set (class I) was spiked with five isotope-labeled compounds, whereas another set (class II) was spiked with six different isotope-labeled compounds. In study II, focus was also on comparing two sets of plasma samples, however, the isotope-labeled compounds were spiked to both class I and class II samples but with concentrations which differ by a factor two between both classes (with one compound absent in each class). The aim was to determine whether CEMS-based metabolomics could reveal the spiked biomarkers as the main classifiers, applying two different data analysis software tools (MetaboAnalyst and Matlab). Unsupervised analysis of the recorded metabolic profiles revealed a clear distinction between class I and class II plasma samples in both studies. This classification was mainly attributed to the spiked isotope-labeled compounds, thereby emphasizing the utility of CE-MS for biomarker discovery. Show less
Electroextraction (EE) and electromembrane extraction (EME) are sample preparation techniques that both require an electric field that is applied over a liquid-liquid system, which enables the... Show moreElectroextraction (EE) and electromembrane extraction (EME) are sample preparation techniques that both require an electric field that is applied over a liquid-liquid system, which enables the migration of charged analytes. Furthermore, both techniques are often used to pre-concentrate analytes prior to analysis. In this review an overview is provided of the body of literature spanning April 2012-November 2015 concerning EE and EME, focused on hyphenation to analytical techniques. First, the theoretical aspects of concentration enhancement in EE and EME are discussed to explain extraction recovery and enrichment factor. Next, overviews are provided of the techniques based on their hyphenation to LC, GC, CE, and direct detection. These overviews cover the compounds and matrices, experimental aspects (i.e. donor volume, acceptor volume, extraction time, extraction voltage, and separation time) and the analytical aspects (i.e. limit of detection, enrichment factor, and extraction recovery). Techniques that were either hyphenated online to analytical techniques or show high potential with respect to online hyphenation are highlighted. Finally, the potential future directions of EE and EME are discussed. Show less
Gulersonmez, M.C.; Lock, S.; Hankemeier, T.; Ramautar, R. 2016
The performance of CE coupled on-line to MS via a sheathless porous tip sprayer was evaluated for anionic metabolic profiling. A representative metabolite mixture and biological samples were used... Show moreThe performance of CE coupled on-line to MS via a sheathless porous tip sprayer was evaluated for anionic metabolic profiling. A representative metabolite mixture and biological samples were used for the evaluation of various analytical parameters, such as peak efficiency (plate numbers), migration time and peak area repeatability, and LODs. The BGE, i.e. 10% acetic acid (pH 2.2), previously used for cationic metabolic profiling was now assessed for anionic metabolic profiling by using MS detection in negative ion mode. For test compounds, RSDs for migration times and peak areas were below 2 and 11%, respectively, and plate numbers ranged from 60 000 to 40 0000 demonstrating a high separation efficiency. Critical metabolites with low or no retention on reversed-phase LC could be efficiently separated and selectively analyzed by the sheathless CE-MS method. An injection volume of only circa 20 nL resulted in LODs between 10 and 200 nM (corresponding to an amount of 0.4-4 fmol), which was an at least tenfold improvement as compared to LODs obtained by conventional CE-MS approaches for these analytes. The methodology was applied to anionic metabolic profiling of glioblastoma cell line extracts. Overall, a sheathless CE-MS method has been developed for highly efficient and sensitive anionic metabolic profiling studies, which can also be used for cationic metabolic profiling studies by only switching the MS detection and separation voltage polarity. Show less
