Spatial profiling with MALDI MS: distribution of neuropeptides within single neurons.
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.
Rubakhin
Stanislav S
SS
Department of Chemistry, Psychology and the Beckman Institute, University of Illinois, Urbana, Illinois 61801, USA.
Greenough
William T
WT
Sweedler
Jonathan V
JV
D000388:Air; D000818:Animals; D001048:Aplysia / Q000737:chemistry / Q000166:cytology; D002478:Cells, Cultured; D003373:Coumaric Acids / Q000737:chemistry; D005466:Fluorocarbons / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D017952:Ganglia, Invertebrate / Q000737:chemistry / Q000166:cytology; D005990:Glycerol / Q000737:chemistry; D016591:Histocytological Preparation Techniques / Q000379:methods; D007447:Invertebrate Hormones / Q000032:analysis; D008853:Microscopy; D008899:Mineral Oil / Q000737:chemistry; D016501:Neurites / Q000737:chemistry; D009474:Neurons / Q000737:chemistry* / Q000166:cytology; D009479:Neuropeptides / Q000032:analysis*; D011108:Polymers / Q000737:chemistry; D012623:Seawater / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014867:Water / Q000737:chemistry
D003373:Coumaric Acids; D005466:Fluorocarbons; D007447:Invertebrate Hormones; D009479:Neuropeptides; D011108:Polymers; C026880:egg-laying hormone, Mollusca; D014867:Water; D005557:Formaldehyde; C007175:alpha-cyano-4-hydroxycinnamate; C008807:perfluorotributylamine; C073734:sinapinic acid; D008899:Mineral Oil; D005990:Glycerol; C003043:paraform