DIRECT PEPTIDE PROFILING BY MASS-SPECTROMETRY OF SINGLE IDENTIFIED NEURONS REVEALS COMPLEX NEUROPEPTIDE-PROCESSING PATTERN

A novel strategy combining peptide fingerprinting of single neurons by matrix-assisted laser desorption ionization mass spectrometry, molecu lar cloning, peptide chemistry, and electrospray ionization mass spect rometry was used to study the intricate processing pattern of a prepro hormone expressed in identified neurons, the neuroendocrine light yell ow cells (LYCs) of the gastropod mollusc, Lymnaea stagnalis. The cDNA encoding the precursor, named prepro-LYCP (LYCPs, light yellow cell pe ptides), predicts a straightforward processing into three peptides, LY CP I, II, and III, at conventional dibasic processing sites flanking t he peptide domains on the precursor. However, matrix-assisted laser de sorption ionization mass spectrometry of single LYCs revealed trimmed variant peptides derived from LYCP I and II. The variants were much mo re abundant than the intact peptides, indicating that LYCP I and II se rve as intermediates in a peptide-processing sequence. Using the molec ular masses of the peptides as markers to guide their isolation by wel l established purification methods, the structural identities of the p eptides could be confirmed by amino acid sequencing. Furthermore, matr ix-assisted laser desorption ionization mass spectrometry could detect colocalization of a novel peptide with the LYCPs.