Combination of snap freezing, differential pH two-dimensional reverse-phase high-performance liquid chromatography, and iTRAQ technology for the peptidomic analysis of the effect of prolyl oligopeptidase inhibition in the rat brain

In vitro, prolyl oligopeptidase (POP) cleaves proline-containing bioactive peptides such as substance P, gonadotropin-releasing hormone, thyrotropin-releasing hormone, arginine-vasopressin, and neurotensin. Based on specific in vivo inhibition, POP has been suggested to be involved in cognitive and psychiatric processes but the identity of its physiological substrates has remained inconclusive. We have combined (a) sample snap-freezing and boiling buffer extraction, to limit protein degradation and reduce sample complexity; (b) pH two-dimensional liquid reverse-phase chromatography to enhance resolution; and (c) iTRAQ isobaric labeling to identify the rat brain peptides whose levels were differentially changed due to in vivo POP inhibition. In the hypothalamus, all the substrates found were part of precursors of secreted peptides such as copeptin, PACAP-related peptide, somatostatin, and proSAAS derived peptides, while in the cerebellum the peptides were derived from carcinoma-amplified sequence 1 homolog and calmodulin. In the striatum, somatostatin precursor derived peptide, fragments from E3-SUMO protein ligase RanBP2, and the subunit 5A of cytochrome c oxidase were increased. When analyzing the peptides that were significantly reduced by POP inhibition we found fragments from large protein complexes but, exclusively in the cerebellum, bioactive peptides such as cerebellin and fibrinopeptides A and B were detected.