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The proteome and peptidome are defined as the set of proteins and peptides present in a tissue or other biological sample. In most proteomic studies, only abundant proteins are detected and, although these are important molecules, they are often well-studied structural proteins. A number of approaches have been used to examine less abundant molecules that play roles in signaling or otherwise have regulatory functions, including peptides as well as proteins such as enzymes and receptors. The overarching goals of this special issue involve defining the peptidome, identifying the current state of the field, and discussing methods to characterize the peptides, their receptors, and future needs for such measurements.

Metallocarboxypeptidase D (CPD) functions in protein and peptide processing. The Drosophila CPD svr gene undergoes alternative splicing, producing forms containing 1-3 active or inactive CP domains. To investigate the function of the various CP domains, we created transgenic flies expressing specific forms of CPD in the embryonic-lethal svr (PG33) mutant. All constructs containing an active CP domain rescued the lethality with varying degrees, and full viability required inactive CP domain-3. Transgenic flies overexpressing active CP domain-1 or -2 were similar to each other and to the viable svr mutants, with pointed wing shape, enhanced ethanol sensitivity, and decreased cold sensitivity. The transgenes fully compensated for a long-term memory deficit observed in the viable svr mutants. Overexpression of CP domain-1 or -2 reduced the levels of Lys/Arg-extended adipokinetic hormone intermediates. These findings suggest that CPD domains-1 and -2 have largely redundant functions in the processing of growth factors, hormones, and neuropeptides.

We recently developed a quantitative peptidomics method using stable isotopic labels and mass spectrometry to both quantify and identify a large number of peptides. To test this approach and screen for peptides regulated by cocaine administration, 32 Cpefat/fat mice and 16 wild-type mice were treated twice daily for 5 d either with saline or 10 mg/kg cocaine. Peptides were extracted from striatum, hypothalamus, hippocampus, and prefrontal cortex, and extracts from groups of eight mice were labeled with the N-hydroxysuccinimide ester of trimethylammonium butyrate containing either nine deuterium or nine hydrogen atoms. Pools of heavy- and light-labeled peptides were combined, purified on an anhydrotrypsin affinity column, and analyzed on a reversephase column coupled to an electrospray ionization quadrapole time-of-flight mass spectrometer. Changes in peptide levels upon cocaine treatment were determined from the relative peak intensities of the cocaine versus saline peaks, and peptides were identified from collision-induced dissociation spectra. Ten peptides were found to increase or decrease in each of two separate analyses from distinct groups of mice. Peptides found to increase corresponded to fragments of proenkephalin, prothyrotropin-releasing hormone, provasopressin, proSAAS, secretogranin II, chromogranin B, and peptidyl-glycine-alpha-amidating mono-oxygenase in the hypothalamus. The same peptidyl-glycine-alpha-amidating mono-oxygenase peptide decreased in the prefrontal cortex, along with striatal neurokinin B and two unidentified peptides. Thirty other peptides were not substantially affected by cocaine treatment in both replicates. Taken together, the quantitative peptidomics approach provides an efficient method to screen for changes in a large number of peptides.

Chronic morphine administration is known to affect several neuropeptide systems, and this could contribute to the behavioral effects of opiates. To quantitate global changes in neuropeptide levels upon chronic morphine administration, we took advantage of a method that allows selective isolation of neuropeptides from brains of mice lacking carboxypeptidase E (Cpefat/fat mice), a critical enzyme in the generation of many neuroendocrine peptides. We used a differential labeling procedure with stable isotopic tags and mass spectrometry to quantitate the relative changes in a number of hypothalamic and striatal peptides in Cpefat/fat mice chronically treated with morphine. A total of 27 distinct peptides were detected in hypothalamus and striatum. Of these, 27 were identified by mass spectrometry-based sequencing, 1 was tentatively identified by the mass and charge, and 9 were not identified. The identified peptides included fragments of proenkephalin, prothyrotropin-releasing hormone, secretogranin II, chromogranin A and B, protachykinin B, provasopressin, promelanin concentrating hormone, and pro-SAAS. Upon morphine administration, although the levels of most of the peptides were unaltered (within a factor of 1.3 to 0.7 compared with saline control), the levels of a small number of peptides did show consistent changes (increased or decreased by 1.3-fold or more) in hypothalamus and/or striatum. Taken together, these results provide interesting insights into endogenous neuropeptide systems that are modulated by morphine and suggest further experiments to link candidate peptides with long-term effects of morphine.