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 PC-mediated 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 alpha-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.

Fig. 1.

Gel filtration profiles of culture supernatant extracts from TT cells before (black trace) and after stimulation (gray trace). Arrows indicate molecular mass markers: A, 66,500 Da; B, 4,271 Da; C, 1,673 Da; D, 556 Da.

Fig. 2.

The numbers of peptides identified before and after stimulation sorted by precursor names. CT and CGRP are grouped as they arise from alternatively spliced exons. Peptide sequences are indicated in Table I. PACAP, pituitary adenylate cyclase-activating polypeptide; NPW, neuropeptide W; PENK, proenkephalin A; KRT, cytokeratin; TMSB4X, thymosin β-4 X-linked; TMSB10, thymosin β-10.

Fig. 3.

Representative base peak chromatograms of the secretopeptidome from unstimulated (top) and stimulated (bottom) cells. Samples without gel filtration chromatography were analyzed. Major processing products of CT and CGRP precursors are illustrated along with arrows pointing at their peaks in the chromatogram. The base peaks marked with asterisks at 20.85 min (unstimulated) and 21.50 min (stimulated) are intact ubiquitin. SP, signal peptide; N-term pro., N-terminal propeptide; Kat., katacalcin. Base peaks at 8.75 and 15.03 min in the top panel were unrelated to peptide signals. RT, retention time; NL, normalized ion intensity.

Fig. 4.

Pie representation of the 400 peptides (sorted by names of 23 precursors) secreted after stimulation. PACAP, pituitary adenylate cyclase-activating polypeptide; NPW, neuropeptide W; PENK, proenkephalin A; KRT, cytokeratin; TMSB4X, thymosin β-4 X-linked; TMSB10, thymosin β-10; PAM, peptidyl-glycine α-amidating monooxygenase; NMU, neuromedin U.

Fig. 5.

CT precursor processing deduced by a panel of identified peptides. The established signal peptide cleavage site (open arrowhead) and known processing sites (closed arrowheads) are shown across the top of the CT precursor with basic residues (thin black boxes) and the signal peptide (hatched box) indicated. Sequences of identified peptides (bars) are detailed in Tables I and II. Major peptides, defined in Table II, are indicated by gray bars. Black boxes denote C-terminally amidated residues.