Ultrasensitive Single Cell Metabolomics by Capillary Electrophoresis–Mass Spectrometry with a Thin-Walled Tapered Emitter and Large-Volume Dual Sample Preconcentration

Abstract

Single cell metabolome analysis is essential for studying microscale life phenomena such as neuronal networks and tumor microenvironments. Capillary electrophoresis−mass spectrometry (CE-MS) is one of the most sensitive technologies; however, its sensitivity is still not enough for single cell analysis on general human cells such as HeLa. To address these issues, we first developed an efficient ionization emitter, named as a "nanoCESI" emitter, that had a thin-walled (∼10 μm) and tapered (5−10 μm) end. The thin conductive wall enabled sheathless ionization and minimized the flow rate of ionizing sample, and the tapered end efficiently ionized analytes via an electrospray ionization mechanism, providing up to 3.5-fold increase in sensitivity compared with a conventional sheathless emitter. Fifty repetitive analyses on 20 amino acids were successfully achieved with a nanoCESI emitter. Relative standard deviations of 50 analyses were 1.5%, 4.4%, and 6.8% for migration time, peak height, and peak area, respectively, where a limit of detection (LOD) of 170 pM (850 zmol) was achieved. Second, a sample enrichment method, large-volume dual preconcentration by isotachophoresis and stacking (LDIS), was applied to a newly designed protocol of nanoCESI-MS. This approach achieved up to 380-fold enhanced sensitivity and LOD of 450 fM. Compared with normal sheathless CE-MS, coupling of nanoCESI and LDIS provided up to 800-fold increase of sensitivity in total. Finally, metabolome analyses of single HeLa cells were performed, where 20 amino acids were successfully quantified with triplequadrupole MS and 40 metabolites were identified with quadrupole-time-of-flight MS, as a promising analytical platform for microscale bioanalysis for the next generation.

CE‐MS for metabolomics: Developments and applications in the period 2018–2020

“…Another aspect that can contribute to single cell metabolomics studies is by optimizing CE‐MS interfacing techniques to achieve more efficient ionization, which in combination with preconcentration techniques will greatly improve the capability of CE‐MS in handling single cells. To enable adequate sensitivity in metabolic profiling of single human cells, Kawai et al [29] incorporated a “nanocapillary electrophoresis with sheathless interface (CESI)” emitter with a large‐volume dual preconcentration technique. The fabrication of a “nanoCESI” emitter utilized fused‐silica capillary (internal diameter/outer diameter [ID/OD], 50 µm/360 µm), with the polyimide at one end etched away in hydrofluoric acid (HF) till the wall thickness reduced to 20–30 µm.…”

Resolving Metabolic Heterogeneity in Experimental Models of the Tumor Microenvironment from a Stable Isotope Resolved Metabolomics Perspective

“…For metabolomic analysis, this remains challenging because of the low abundance of most metabolites in each cell and the difficulty of maintaining biochemical integrity and minimizing metabolite diffusion during cell isolation or in situ analysis [55]. For example, a metabolite present at 1 µM in a cell of 1 pL volume is only one attomole of material, which while achievable [167] is challenging to quantify even by the best mass spectrometers under ideal conditions, and is well beyond the capabilities of NMR [168] even with hyperpolarization [169]. To the best of our knowledge, single cell tracer studies have not been reported, presumably due to detectability issue.…”

Profiling nucleotides in low numbers of mammalian cells by sheathless CE–MS in positive ion mode: Circumventing corona discharge

“…CZE is a highly efficient microscale separation technique and as compounds are separated on the basis of their charge‐to‐size ratio, CZE is especially suited for the profiling of polar and charged compounds under biocompatible conditions . Moreover, CZE–MS is well‐suited for the sensitive analysis of compounds in material‐ or volume‐limited samples as only nanoliter injection volumes are required from just a few microliters of sample or less in the injection vial . A number of recent studies have clearly shown the utility of CZE–MS for metabolic profiling of large sample sets.…”