Snapshot Peptidomics of the Regulated Secretory Pathway

Abstract

Neurons and endocrine cells have the regulated secretory pathway (RSP) in which precursor proteins undergo proteolytic processing by prohormone convertase (PC) 1/3 or 2 to generate bioactive peptides. Although motifs for PCmediated processing have been described ((R/K)X n (R/K) where n ‫؍‬ 0, 2, 4, or 6), actual processing sites cannot be predicted from amino acid sequences alone. We hypothesized that discovery of bioactive peptides would be facilitated by experimentally identifying signal peptide cleavage sites and processing sites. However, in vivo and in vitro peptide degradation, which is widely recognized in peptidomics, often hampers processing site determination. To obtain sequence information about peptides generated in the RSP on a large scale, we applied a brief exocytotic stimulus (2 min) to cultured endocrine cells and analyzed peptides released into supernatant using LC-MSMS. Of note, 387 of the 400 identified peptides arose from 19 precursor proteins known to be processed in the RSP, including nine peptide hormone and neuropeptide precursors, seven granin-like proteins, and three processing enzymes (PC1/3, PC2, and peptidyl-glycine ␣-amidating monooxygenase). In total, 373 peptides were informative enough to predict processing sites in that they have signal sequence cleavage sites, PC consensus sites, or monobasic cleavage sites. Several monobasic cleavage sites identified here were previously proved to be generated by PCs. Thus, our approach helps to predict processing sites of RSP precursor proteins and will expedite the identification of unknown bioactive peptides hidden in precursor sequences.

Large-scale Identification of Endogenous Secretory Peptides Using Electron Transfer Dissociation Mass Spectrometry

“…Previously, a limited number of VGF-derived peptides were reported in peptidomics work on human cerebrospinal fluid using CID (20 -22). We conducted a more detailed study by analyzing the culture supernatant from a human endocrine cell line and tissue extracts in normal rat brain and gut (3,4,23). In earlier work, several CgB-derived peptides have been isolated from extracts of human pheochromocytoma and neuroendocrine tumors (24 -26).…”

“…Owing to their compartmentalization, secretory peptides can be noninvasively recovered in culture supernatant. We have shown that a data set of endogenous peptide sequences that are collected by this procedure is applicable to infer processing sites, as well as to identify bona fide processing products (4). Rather than being digested, every endogenous peptide should be analyzed in its native form to understand how the peptide is generated and subsequently degraded.…”

Quantitative Peptidomics Study Reveals That a Wound-Induced Peptide from PR-1 Regulates Immune Signaling in Tomato

“…In animal tissues, peptidomics approaches have been used to elucidate novel bioactive peptides and their functions (Svensson et al, 2003;Che et al, 2005;Sasaki et al, 2009;Tinoco and Saghatelian, 2011). However, very few successful cases of discovery of plant signaling peptides have been reported.…”

Simultaneous quantification of intracellular and secreted active and inactive glucagon-like peptide-1 from cultured cells

“…The peptide fraction was collected using a reverse-phase extraction cartridge (InertSep RP-1, GL Sciences) and analyzed by nano-LC-MS/MS, as described by Sasaki and coworkers [26,27].…”