EdwardJ.Bures 1 , PaulL.Courchesne 2 , JamesDouglass 3 , KuiChen 2 , MichaelT.Davis 1 , MichaelD.Jones 1 , MichaelD.McGinley 1 , JohnH.Robinson 1 , ChrisS.Spahr 1 , JilinSun 3 , RobertC.Wahl 2 , ScottD.Patterson 1 Departments of Biochemistry and Genetics 1 , HTS Molecular Pharmacology 2 and Neurobiology 3 , Amgen, Thousand Oaks, CA, USA Identificationofincompletelyprocessedpotential CarboxypeptidaseEsubstratesfromCpE fat /CpE fat mice In an attempt to identify peptides that may be involved in the obese phenotype observed in CpE fat /CpE fat mice (deficient in Carboxypeptidase E, CpE) samples from fourteen neuroendocrine tissues in wild-type and CpE fat /CpE fat mice were obtained. Peptides were purified from these tissues and potential CpE substrate peptides were enriched using an anhydrotrypsin column that captures peptides with basic C-ter- mini. Bound peptides were subjected to tryptic digestion and followed by liquid chro- matography-mass spectrometry analysis. The relative levels of CpE fat /CpE fat versus wild-type peptides were determined by comparison of the ion intensities. Peptide ions elevated in the CpE fat /CpE fat samples were identified by targeted liquid chroma- tography-tandem mass spectrometry. From those ions, 27 peptides derived from known neuropeptides (including CpE substrates) were identified, together with an- other 25 peptides from proteins not known to be components of the neuropeptide processing pathway. The known CpE substrates identified included the recently dis- covered proSAAS, granin-like neuroendocrine peptide precursor that inhibits prohor- mone processing. The approach demonstrated the feasibility of using an affinity- based method for identifying differences in specific classes of peptides between nor- mal and mutant mice. Keywords: Liquid chromatography-mass spectrometry / Proteomics / Proneuropeptides / Fat mice PRO 0016 1 Introduction Mature peptide hormones are usually generated from enzyme directed processes that proteolytically modify larger precursor propeptides [1]. An interruption of the biosynthetic pathway may occur if one of the requisite enzymes is deficient, resulting in the build-up of uncon- verted intermediate species along with the absence of the final mature product hormone. For example, as shown in Fig. 1 in the biosynthesis of secretogranin II, a signal peptidase removes the signal peptide, a serine protease proprotein convertase then cleaves at multiple dibasic Arg-Arg or LysArg residues to form intermedia- tes. These intermediates are substrates for Carboxypep- Figure1. Biosynthetic processing of secretogranin II pre- cursor. A schematic showing three of the common featu- res of proneuropeptide processing, signal peptide remo- val, proprotein convertase cleavage at dibasic residues, and carboxypeptidase removal of the exposed dibasic residues. Correspondence: Dr. Scott D. Patterson, Celera Genomics, 45 West Gude Drive, Rockville, MD 20850, USA E-mail: scott.patterson@celera.com Fax: +1-240-453-4666 Abbreviations: amu, atomic mass unit; CpE, carboxypeptidase E; m/z, mass-to-charge ratio Proteomics 2001, 1, 79–92 79  WILEY-VCH Verlag GmbH, 69451 Weinheim, 2001 1615-9853/01/0101–0079 $17.50+ .50/0