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 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-alpha-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.
Jespersen
S
S
Department of Bio-Pharmaceutical Analysis, TNO Pharma, AJ Zeist, The Netherlands. jespersen@voeding.tno.nl
Chaurand
P
P
van Strien
F J
FJ
Spengler
B
B
van der Greef
J
J
D000595:Amino Acid Sequence; D000818:Animals; D009479:Neuropeptides / Q000032:analysis*; D010902:Pituitary Gland / Q000737:chemistry; D017421:Sequence Analysis / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014982:Xenopus laevis