Metabolic Differentiation of Neuronal Phenotypes by Single-cell Capillary Electrophoresis–Electrospray Ionization-Mass Spectrometry

Abstract

Single cell mass spectrometry (MS) is a rapidly emerging field in metabolic investigations. The inherent chemical complexity of most biological samples poses analytical challenges when using MS platforms to measure sample content without prior chemical separation. Here, a single-cell capillary electrophoresis (CE) system was coupled with electrospray ionization (ESI) MS to enable the simultaneous measurement of a vast array of endogenous compounds in over 50 identified and isolated large neurons from the Aplysia californica central nervous system. More than 300 distinct ion signals (m/z values) were detected from a single neuron in positive ion mode, 140 of which were selected for chemometric data analysis. Metabolic features were evaluated among six different neuron types (B1, B2, left pleural 1 (LPl1), metacerebral cell (MCC), R2, and R15), chosen for their various physiological functions. The results indicated chemical similarities among some neuron types (B1 to B2 and LPl1 to R2) and distinctive features for others (MCC and R15 cells). The quantitative nature of the MS platform allowed the comparison of metabolite levels for specific neurons. The CE-ESI-MS approach for examination of individual nanoliter-volume cells as described herein is readily adaptable to other volume-limited samples.

Label-free Quantification of Proteins in Single Embryonic Cells with Neural Fate in the Cleavage-Stage Frog (Xenopus laevis) Embryo using Capillary Electrophoresis Electrospray Ionization High-Resolution Mass Spectrometry (CE-ESI-HRMS)

“…Electrophoresis is a rapidly emerging alternative technology for highsensitivity proteomics. Electrophoretic separation affords exquisite peak capacity, is compatible with diverse types of molecules, can be hyphenated to ESI-HRMS via various interface designs, and is scalable to single cells (22)(23)(24)(25)(26)(27). Using microscale chemical separation to simplify sample chemistry, capillary electrophoresis (CE), and Fourier transform MS was used to target ␣-and ␤-globulins in 5-10 human erythrocytes (28) and carbonic anhydrase (23) in lysates diluted to single cells.…”

Small-Volume Analysis of Cell–Cell Signaling Molecules in the Brain

“…In a study combining CE separation, MS identification, and chemometric quantification, we investigated cellular heterogeneity among physiologically well-characterized identified neurons of A. californica. Out of 300 detected species, 144 were structurally identified, and 50 quantified, revealing surprising differences, not only between neuronal types but also among individual neurons of the same type (Nemes et al, 2011). These observations contribute new evidence about the physiological differences in neuron phenotypes.…”

“…CE-MS (see Figure 3a) is widely used to measure and identify bioactive peptides (Ye et al, 2011), metabolites (Nemes et al, 2011(Nemes et al, , 2012Nautiyal et al, 2012;Gholipour et al, 2013), classical neurotransmitters (Lapainis et al, 2009), and amino acids (Moini, 2013). Alternatively, microdialysis sampling can be directly coupled to ESI-MS via nanodroplet segmented flow for in vivo chemical monitoring of neurotransmitters, metabolites, and drugs in the live brain (Song et al, 2012).…”

Mass spectrometry-based characterization of endogenous peptides and metabolites in small volume samples

“…These techniques are often used to obtain key structural information on detected metabolites and peptides, including post-translational modifications [5], and can be applied in small volume analysis. CE-MS and CE-MS/MS demonstrated good performance in the investigation of a single cell metabolome [81], with more than 300 molecular features detected in a single neuron and 30 metabolite identities confirmed. The approach allows different types of neurons and their functional conditions/states to be distinguished [82].…”