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

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 reveal ed both previously known and unexpected processing products of the proopiom elanocortin gene. Mass spectrometric peptide sequencing of a few of these n europeptides was performed by employing MALDI combined with postsource deca y (PSD) fragment ion mass analysis. The potential of MALDI-PSD for sequence analysis of peptides directly from unfractionated tissue samples was exami ned for the first time for the known desacetyl-alpha-MSH-NH2 and the presum ed 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 containe d sufficient structural information to uniquely identify the sequence by se arching protein sequence databases. The determined amino acid sequence corr esponds to the vasotocin peptide with a C-terminal extension of Gly-Lys-Arg ("vasotocinyl-GKR"), indicating incomplete processing of the vasotocin pre cursor protein in the pituitary pars intermediate of X. laevis. Both vasoto cin and vasotocinyl-GKR are nonlinear peptides containing a disulfide (S-S) bridge between two cysteine residues. Interpretation of the spectra of the se two peptides reveals three different forms of characteristic fragment io ns of the cysteine side chain: peptide-CH2-SH (regular mass of Cys-containi ng 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.