Chiral Measurement of Aspartate and Glutamate in Single Neurons by Large-Volume Sample Stacking Capillary Electrophoresis

Abstract

D-Amino acids (D-AAs) are endogenous molecules found throughout the metazoan, the functions of which remain poorly understood. Measurements of low abundance and heterogeneously distributed D-AAs in complex biological samples, such as cells and multicellular structures of the central nervous system (CNS), require the implementation of sensitive and selective analytical approaches. In order to measure the D- and L-forms of aspartate and glutamate, we developed and applied a stacking chiral capillary electrophoresis (CE) with laser-induced fluorescence detection method. The achieved online analyte preconcentration led to a 480-fold enhancement of detection sensitivity relative to capillary zone electrophoresis, without impacting separation resolution or analysis time. Additionally, the effects of inorganic ions on sample preconcentration and CE separation were evaluated. The approach enabled the relative quantification of D-aspartate and D-glutamate in individual neurons mechanically isolated from the CNS of the sea slug Aplysia californica, a well-characterized neurobiological model. Levels of these structurally similar D-AAs were significantly different in sub-populations of cells collected from the investigated neuronal clusters.

Recent progress of sample stacking in capillary electrophoresis (2016–2018)

“…Patel et al. investigated low‐abundance and heterogeneously distributed D‐amino acids in complex biological samples, such as cells and multicellular structures of the central nervous system. The developed LVSS‐chiral CE method with LIF detection for the analysis of D‐ and L‐forms of aspartate and glutamate uses a BGE comprising 60 mM MES and 10 mM KBr at pH 6 with 110 ppm quaternary ammonium β‐CD.…”

Exploring the Fundamental Structures of Life: Non‐Targeted, Chemical Analysis of Single Cells and Subcellular Structures

“…TheS weedler group incorporated sample stacking with single-cell CE-LIF to allow chiral separations of the d-a nd l-forms of aspartate and glutamate within individual Aplysia neurons. [39] Theseparation power and large dynamic range of CE allowed statistically distinct levels of the d-amino acids to be quantitated within different cell types.C E-LIF has also been used to study proteins within individual cancer cells, discovering heterogeneity within the expression of cysteine cathepsins. [40] While single cells are the smallest functional unit of life, subcellular organelles actively manage and participate in hierarchical organization, expression, and intercellular communication.…”

Recent advances in enhancing the sensitivity of electrophoresis and electrochromatography in capillaries and microchips (2016–2018)

“…In LVSEP, the EOF that is smaller and opposite to the electrophoretic mobility of the analytes is used to pump the sample matrix out of the capillary. LVSEP has been used for sensitive and efficient determination of nitrate and nitrite in canned fish samples ; d ‐ and l ‐ isomers of aspartate and glutamate with enrichment factor of 480 ; anti‐microbial agent, pentamidine in rat plasma ; ammonia, amines (including cyclohexylamine, ethanolamine, morpholine, hydrazine, dimethylamine, and triethanolamine) and their degradation products, e.g., methylamine, ethylamine, and diethanolamine in steam water ; and DNA fragments . Multistep pressure assisted LVSEP, in which injection of large sample volumes and pressure assisted electroosmotic pumping of the sample matrix out of the capillary was performed in repeated cycles to improve the sensitivity (enhancement factors up to 170) of amyloid β (Aβ) peptides, biomarkers for Alzheimer's disease diagnosis, in cerebrospinal fluid .…”