Mass Spectrometry-Based Neuropeptidomics of Secretory Vesicles from Human Adrenal Medullary Pheochromocytoma Reveals Novel Peptide Products of Prohormone Processing

Abstract

Neuropeptides are required for cell-cell communication for the regulation of physiological and pathological processes. While selected neuropeptides of known biological activities have been studied, global analyses of the endogenous profile of human peptide products derived from prohormones by proteolytic processing in vivo is largely unknown. Therefore, this study utilized the global, unbiased approach of mass spectrometry-based neuropeptidomics to define peptide profiles in secretory vesicles, isolated from human adrenal medullary pheochromocytoma of the sympathetic nervous system. The low molecular weight pool of secretory vesicle peptides was subjected to nano-LC-MS/MS with ion trap and QTOF mass spectrometry analyzed by different database search tools (InsPecT and Spectrum Mill). Peptides were generated by processing of prohormones at dibasic cleavage sites as well as at non-basic residues. Significantly, peptide profiling provided novel insight into newly identified peptide products derived from proenkephalin, pro-NPY, proSAAS, CgA, CgB, and SCG2 prohormones. Previously unidentified intervening peptide domains of prohormones were observed, thus, providing new knowledge of human neuropeptidomes generated from precursors. The global peptidomic approach of this study demonstrates the complexity of diverse neuropeptides present in human secretory vesicles for cellcell communication.

Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters

“…For analysis of peptide products by nano-LC-MS/MS tandem mass spectrometry, samples in 1% formic acid were analyzed by the nano-LC-MS/MS XCT Ultra ion trap mass spectrometer instrument (Agilent Technologies, Inc., Wilmington, DE) as we have described previously (22). Peptides were identified using Spectrum Mill or InsPecT software with the search of both the human RefSeq data base (NCBI) and a data base of the human PE sequence.…”

“…The fresh pheochromocytoma tissue was used for purification of secretory vesicles (also known as chromaffin granules, CG) by differential sucrose density gradient centrifugation, as we have previously described (22). The high purity of these secretory vesicles, achieved by sucrose density gradient centrifugation, has been documented by enzyme markers showing the lack of other organelle markers in the purified secretory vesicle preparation and by electron microscopy showing the purity, homogeneity, and integrity of the purified secretory vesicles (23)(24)(25)(26).…”

Pyroglutamate-Amyloid-β and Glutaminyl Cyclase Are Colocalized with Amyloid-β in Secretory Vesicles and Undergo Activity-Dependent, Regulated Secretion

“…An acid extract (0.1 N acetic acid) was prepared from the lysed secretory vesicles (as described previously [25, 26, 34, 35]) for measurement of pGlu-Aβ(3-40), pGlu-Aβ(3-42), Aβ(1-40), Aβ(1-42), (Met)enkephalin, and galanin, and the catecholamines dopamine, norepinephrine, and epinephrine. DCSV samples were subjected to ELISA measurements of pGlu-Aβ(3-40) (#27418, IBL International, Toronto, Canada), pGlu-Aβ(3-42) (#27716, IBL), Aβ(1-40) (#27718, IBL), Aβ(1-42) (#27712, IBL), with RIA assays for (Met)enkephalin and galanin (RIAs were conducted as previously reported [28, 33, 35].…”

Neuropeptidomics Mass Spectrometry Reveals Signaling Networks Generated by Distinct Protease Pathways in Human Systems

“…Neuropeptidomics analyses by nano-liquid chromatography tandem mass spectrometry (LC-MS/MS) of human secretory vesicles (isolated from human pheochromocytoma) is capable of revealing a repertoire of processed peptides derived from a single pro-neuropeptide [12]. Furthermore, LC-MS/MS in one experiment provides data for the spectrum of peptide products derived from multiple pro-neuropeptides.…”