Spatial Profiling with MALDI MS:  Distribution of Neuropeptides within Single Neurons

Abstract

MALDI MS imaging and single-cell profiling are important new capabilities for mass spectrometry. The distribution of neuropeptides within a cell plays an important role in the functioning of the cells in a neuronal network. Protocols for subcellular MALDI MS are described that allow comparative peptide profiling of cell bodies and the neuronal processes (neurites) using single isolated neurons from the neuronal model Aplysia californica. The seawater surrounding the neurons is problematic for mass spectrometry and so must be removed in a manner that does not cause morphological changes or a redistribution of the neuropeptides. Several protocols have been investigated for subcellular spatial profiling, including the use of air-drying, replacement of the seawater with deionized water, and substitution of the cell matrix with fluorinert, mineral oil and glycerol, as well as paraformaldehyde fixation. Glycerol stabilization offers the best combination of preservation of cell morphology and prevention of neuropeptide redistribution. The profiles of the peptides in specific neuronal processes and the cell bodies demonstrate a variety of differences that appear to be cell-specific. These methods are suitable for smaller cells and subcellular MS imaging.

Neuropeptidomics: MS Applied to the Discovery of Novel Peptides from the Brain

“…This procedure is commonly used in immunohistochemical methods to detect neurotransmitters and neuromodulators and in individual neurites to produce excellent spatial information (41). However, this procedure reduces the intensity of the peptide peaks in MS, probably because many peptides become crosslinked into the polymer matrix by fixation (42).…”

In-situ probing of the biotic-abiotic boundary of plants by laser desorption/ionization time-of-flight mass spectrometry

“…For example, MALDI-TOF has been recently used for the direct profiling of peptides and proteins in single cells or tissues [15][16][17][18]. However, since LDI-TOF is primarily a surfaceprobing tool, e.g., ions are generated only from the exterior regions of the sample that are exposed to laser irradiation [19], determination of intracellular contents usually requires special sample preparation [15].…”

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

“…For example, sampling of live individual neurons and subcellular neural regions from ganglia, nerves, or other anatomical structures is carried out primarily by hand. In our group, hundreds of samples of individual, live invertebrate, and mammalian nerve cells, and even individual dense core vesicles, have been manually isolated under stereomicroscopic observation from intact ganglia with sharpened tungsten carbide dissection needles (Fuller et al, 1998;Rubakhin et al, 2003Rubakhin et al, , 2006Romanova et al, 2004Romanova et al, , 2007Romanova et al, , 2012Ye et al, 2008). In another approach, individual cells or miniature defined morphological regions have been removed from frozen or fixed tissue sections for neuropeptide analysis .…”