Single-cell Metabolic Profiling of the Aplysia californica Nervous System by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry

摘要

The cell-to-cell variation within the nervous system is enormous, with this variation representing physiological, chemical, and functional differences; single-cell mass spectrometry (MS) is an information-rich bioanalytical approach well-suited to probing these chemical differences within neurons. Among the various ionization platforms for MS, matrix-assisted laser desorption/ionization and secondary ion mass spectrometry have shown considerable success in cellular and sub-cellular measurements. Here, we hyphenate chemical separation with MS detection for the metabolic characterization of individual neurons from the marine slug Aplysia californica. Capillary electrophoresis (CE) followed by electrospray ionization (ESI) ensures efficient liquid-phase separation and ion generation of endogenous metabolites. Principal component analysis (PCA) identifies characteristic metabolic patterns of selected neurons, including the metacerebral (MCC) and R2 cells. Ganglia and adjacent nerves of three adult A. californica were surgically dissected and selected neurons were manually isolated. The cells were placed into 50percent (v/v) aqueous methanol solution containing 0.1percent (v/v) formic acid for analyte extraction. Aliquots of the resulting solution (500 nL) were transferred into a sample loading vial and a volume equal to approx0.1percent of the total cell content (approx6 nL) was hydrodynamically injected into a fused silica capillary (90-100 cm in length). Electrophoretic separation was performed in 1percent aqueous formic acid solution at 20 kV potential. Eluting compounds entered a custom-built sheath-flow electrospray interface operated in cone-jet spraying mode for efficient ion generation as detailed elsewhere. The produced ions were then mass-analyzed by a micrOTOF ESI-TOF-MS or a maXis ESI-Qq-TOF-MS/MS system (Bruker Daltonics; Billerica, MA). Tandem MS experiments with 20-35 eV collision energies facilitated molecular identifications.