Retrograde labeling of single neurons in conjunction with MALDI high-energy collision-induced dissociation MS/MS analysis for peptide profiling and structural characterization.
To reveal the peptide contents of the visually nonidentifiable neurons from a neuronal circuit of interest, we combined retrograde labeling of neurons with mass spectrometric single cell analysis. We used the neuronal circuit involved in the copulation behavior of a freshwater snail, Lymnaea stagnalis, as a model. Central neurons that control this behavior are known to send their axons to the penis nerve and innervate the penis complex. By retrograde filling from the penis nerve with nickel-lysine, these neurons were selectively labeled darkish blue. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometric analyses of single stained neurons in the parietal ganglion from different animals reveal consistently the presence of several molecular ion species in the range of 800-1200 Da. From a single neuron, six molecular ion species were further characterized with MALDI time-of-flight/time-of-flight mass spectrometry, which demonstrates that the peptides are derived from a previously reported -FLRFamide precursor.
El Filali
Zineb
Z
Department of Molecular and Cellular Neurobiology, Faculty of Earth and Life Sciences, Graduate School Neurosciences Amsterdam, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands.
Hornshaw
Martin
M
Smit
August B
AB
Li
Ka Wan
KW
D000595:Amino Acid Sequence; D000818:Animals; D008195:Lymnaea / Q000737:chemistry / Q000166:cytology; D008297:Male; D008969:Molecular Sequence Data; D009474:Neurons / Q000737:chemistry*; D009479:Neuropeptides / Q000032:analysis* / Q000737:chemistry*; D010413:Penis / Q000166:cytology / Q000294:innervation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013194:Staining and Labeling / Q000379:methods
D009479:Neuropeptides