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, molecular cloning, peptide chemistry, and electrospray ionization mass spectrometry was used to study the intricate processing pattern of a preprohormone expressed in identified neurons, the neuroendocrine light yellow cells (LYCs) of the gastropod mollusc, Lymnaea stagnalis. The cDNA encoding the precursor, named prepro-LYCP (LYCPs, light yellow cell peptides), predicts a straightforward processing into three peptides, LYCP I, II, and III, at conventional dibasic processing sites flanking the peptide domains on the precursor. However, matrix-assisted laser desorption ionization mass spectrometry of single LYCs revealed trimmed variant peptides derived from LYCP I and II. The variants were much more abundant than the intact peptides, indicating that LYCP I and II serve as intermediates in a peptide-processing sequence. Using the molecular masses of the peptides as markers to guide their isolation by well established purification methods, the structural identities of the peptides could be confirmed by amino acid sequencing. Furthermore, matrix-assisted laser desorption ionization mass spectrometry could detect colocalization of a novel peptide with the LYCPs.
Li
K W
KW
Graduate School Neurosciences Amsterdam, Research Institute Neurosciences Vrije Universiteit, Faculty of Biology, The Netherlands.
Hoek
R M
RM
Smith
F
F
Jiménez
C R
CR
van der Schors
R C
RC
van Veelen
P A
PA
Chen
S
S
van der Greef
J
J
Parish
D C
DC
Benjamin
P R
PR
D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D017952:Ganglia, Invertebrate / Q000378:metabolism*; D008195:Lymnaea; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism*; D009479:Neuropeptides / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D011498:Protein Precursors / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational
C084575:LYCP-A protein, Lymnaea stagnalis; D009479:Neuropeptides; D010446:Peptide Fragments; D011498:Protein Precursors