Single-Cell Metabolomics: Changes in the Metabolome of Freshly Isolated and Cultured Neurons

Abstract

Metabolites are involved in a diverse range of intracellular processes, including a cell’s response to a changing extracellular environment. Using single-cell capillary electrophoresis coupled to electrospray ionization mass spectrometry, we investigated how placing individual identified neurons in culture affects their metabolic profile. First, glycerol-based cell stabilization was evaluated using metacerebral neurons from Aplysia californica; the measurement error was reduced from ∼24% relative standard deviation to ∼6% for glycerol-stabilized cells compared to those isolated without glycerol stabilization. In order to determine the changes induced by culturing, 14 freshly isolated and 11 overnight-cultured neurons of two metabolically distinct cell types from A. californica, the B1 and B2 buccal neurons, were characterized. Of the more than 300 distinctive cell-related signals detected, 35 compounds were selected for their known biological roles and compared among each measured cell. Unsupervised multivariate and statistical analysis revealed robust metabolic differences between these two identified neuron types. We then compared the changes induced by overnight culturing; metabolite concentrations were distinct for 26 compounds in the cultured B1 cells. In contrast, culturing had less influence on the metabolic profile of the B2 neurons, with only five compounds changing significantly. As a result of these culturing-induced changes, the metabolic composition of the B1 neurons became indistinguishable from the cultured B2 cells. This observation suggests that the two cell types differentially regulate their in vivo or in vitro metabolomes in response to a changing environment.

d-Alanine in the islets of Langerhans of rat pancreas

“…Even with an efficient separation from a complex tissue extract, overlapping peaks can be expected. While mass spectrometry can be used as a detector for CE [40,41,42], its effectiveness for chiral separations requiring surfactant-based chiral selectors is limited. Hence, we used LIF detection to confirm the peak identities via enzymatic degradation, similar to what we used to quantify D-ASP and D-Ser [43,44].…”

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

“…Another benefit of CE, especially for neurochemical applications, is that separation is based on the differential migration of charged molecules; many of the neurotransmitters and neuromodulators in the nervous system are charged over a range of pH values, and thus can be separated from the cellular milieu under typical CE conditions. CE has been successfully used for discovering new serotonin metabolites (Squires et al, 2006), and characterizing the content of single cells (Nemes et al, 2012) and single vesicles (Omiatek et al, 2009). Chiral analysis of excitatory acids from the brain by CE has been reported (Wang et al, 2011;Wagner et al, 2012).…”

“…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).…”

“…These observations contribute new evidence about the physiological differences in neuron phenotypes. In a follow-up study, we explored how culturing conditions affect the metabolic profile of individual neurons and found statistically significant changes in the levels of amino acids and small transmitters linked to cell culture conditions (see Figure 3c) (Nemes et al, 2012). With proper experimental design, small-volume exploratory measurements of biological samples may lead to the discovery of biomarkers for disease states.…”

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

“…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]. …”