Identification of peptides from brain and pituitary of Cpe(fat)/Cpe(fat) mice

Cpe(fat)/Cpe(fat) mice have a naturally occurring point mutation within the carboxypeptidase E gene that inactivates this enzyme, leading to an accumu lation of many neuroendocrine peptides containing C-terminal basic residues . These processing intermediates can be readily purified on an anhydrotryps in affinity resin. Using MS to obtain molecular mass and partial sequence i nformation, more than 100 peptides have been identified. These peptides rep resent fragments of 16 known secretory pathway proteins, including proenkep halin, proopiomelanocortin, protachykinins A and B, chromogranin A and B, a nd secretogranin Il. Many of the identified peptides represent previously u ncharacterized fragments of the precursors. For example, 12 of the 13 chrom ogranin B-derived peptides found in the present study have not been previou sly reported. Of these 13 chromogranin B-derived peptides, only five contai n consensus cleavage sites for prohormone convertases at both the C and N t ermini. Two distinct chromogranin B-derived peptides result from cleavage a t Trp-Trp bonds, a site not typically associated with neuropeptide processi ng. An RIA was used to confirm that one of these peptides, designated WE-15 , exists in wild-type mouse brain, thus validating the approach to identify peptides in Cpe(fat)/Cpe(fat) mice. These "orphan" peptides are candidate ligands for orphan G protein-coupled receptors. In addition, the general te chnique of using affinity chromatography to isolate endogenous substrates f rom a mutant organism lacking an enzyme should be applicable to a wide rang e of enzyme-substrate systems.