Identification of incompletely processed potential Carboxypeptidase E substrates from CpEfat/CpEfat mice

In an attempt to identify peptides that may be involved in the obese phenot ype observed in CpEfat/CpEfat mice (deficient in Carboxypeptidase E, CpE) s amples from fourteen neuroendocrine tissues in wild-type and CpEfat/CpEfat mice were obtained. Peptides were purified from these tissues and potential CpE substrate peptides were enriched using an anhydrotrypsin column that c aptures peptides with basic C-termini. Bound peptides were subjected to try ptic digestion and followed by liquid chromatography-mass spectrometry anal ysis. The relative levels of CpEfat/CpEfat versus wild-type peptides were d etermined by comparison of the ion intensities. Peptide ions elevated in th e CpEfat/CpEfat samples were identified by targeted liquid chromatography-t andem mass spectrometry. From those ions, 27 peptides derived from known ne uropeptides (including CpE substrates) were identified, together with anoth er 25 peptides from proteins not known to be components of the neuropeptide processing pathway. The known CpE substrates identified included the recen tly dis covered proSAAS, granin-like neuroendocrine peptide precursor that inhibits prohormone processing. The approach demonstrated the feasibility o f using an affinity-based method for identifying differences in specific cl asses of peptides between normal and mutant mice.