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Direct sequencing of neuropeptides in biological tissue by MALDI-PSD mass spectrometry

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Author: Jespersen, S. · Chaurand, P. · Strien, F.J.C. van · Spengler, B. · Greef, J.D. van
Institution: Centraal Instituut voor Voedingsonderzoek TNO
Source:Analytical Chemistry, 3, 71, 660-666
Identifier: 234935
doi: doi:10.1021/ac980841c
Keywords: Nutrition · Neuropeptide · Amino acid sequence · Animal tissue · Desorption · Hypophysis intermediate lobe · Ionization · Mass spectrometry · Nonhuman · Xenopus laevis · Amino Acid Sequence · Animals · Neuropeptides · Pituitary Gland · Sequence Analysis · Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization · Xenopus laevis


Dissected tissue pieces of the pituitary pars intermedia from the amphibian Xenopus laevis was directly subjected to matrix-assisted laser desorption/ionization (MALDI) mass analysis. The obtained MALDI peptide profile revealed both previously known and unexpected processing products of the proopiomelanocortin gene. Mass spectrometric peptide sequencing of a few of these neuropeptides was performed by employing MALDI combined with postsource decay (PSD) fragment ion mass analysis. The potential of MALDI- PSD for sequence analysis of peptides directly from unfractionated tissue samples was examined for the first time for the known desacetyl-α-MSH-NH2 and the presumed vasotocin neuropeptide. In addition, the sequence of an unknown peptide which was present in the pars intermedia tissue sample at mass 1392.7 u was determined. The MALDI-PSD mass spectrum of precursor ion 1392.7 u contained sufficient structural information to uniquely identify the sequence by searching protein sequence databases. The determined amino acid sequence corresponds to the vasotocin peptide with a C-terminal extension of Gly-Lys-Arg ('vasotocinyl-GKR'), indicating incomplete processing of the vasotocin precursor protein in the pituitary pars intermediate of X. laevis. Both vasotocin and vasotocinyl-GKR are nonlinear peptides containing a disulfide (S-S) bridge between two cysteine residues. Interpretation of the spectra of these two peptides reveals three different forms of characteristic fragment ions of the cysteine side chain: peptide-CH2-SH (regular mass of Cys-containing fragment ions), peptide-CH2-S-SH (regular mass + 32 u) and peptide=CH2 (regular mass - 34 u) due to cleavage on either side of the sulfur atoms.