title: C-terminal methylation of truncated neuropeptides: an enzyme-assisted extraction artifact involving methanol.
abstract: Neuropeptides are the largest class of signaling molecules used by nervous systems. Today, neuropeptide discovery commonly involves chemical extraction from a tissue source followed by mass spectrometric characterization. Ideally, the extraction procedure accurately preserves the sequence and any inherent modifications of the native peptides. Here, we present data showing that this is not always true. Specifically, we present evidence showing that, in the lobster Homarus americanus, the orcokinin family members, NFDEIDRSGFG-OMe and SSEDMDRLGFG-OMe, are non-native peptides generated from full-length orcokinin precursors as the result of a highly selective peptide modification (peptide truncation with C-terminal methylation) that occurs during extraction. These peptides were observed by MALDI-FTMS and LC-Q-TOFMS analyses when eyestalk ganglia were extracted in a methanolic solvent, but not when tissues were dissected, co-crystallized with matrix, and analyzed directly with methanol excluded from the sample preparation. The identity of NFDEIDRSGFG-OMe was established using MALDI-FTMS/SORI-CID, LC-Q-TOFMS/MS, and comparison with a peptide standard. Extraction substituting deuterated methanol for methanol confirmed that the latter is the source of the C-terminal methyl group, and MS/MS confirmed the C-terminal localization of the added CD3. Surprisingly, NFDEIDRSGFG-OMe is not produced via a chemical acid-catalyzed esterification. Instead, the methylated peptide appears to result from proteolytic truncation in the presence of methanol, as evidenced by a reduction in conversion with the addition of a protease-inhibitor cocktail; heat effectively eliminated the conversion. This unusual and highly specific extraction-derived peptide conversion exemplifies the need to consider both chemical and biochemical processes that may modify the structure of endogenous neuropeptides.
authors.affiliation: Department of Chemistry, Bowdoin College, 6600 College Station, Brunswick, ME 04011, USA. estemmle@bowdoin.edu
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Stemmler
authors.forename: Elizabeth E
authors.initials: EE
authors.lastname: Barton
authors.forename: Onyinyechi K
authors.initials: OK
authors.lastname: Esonu
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Polasky
authors.forename: Laura L
authors.initials: LL
authors.lastname: Onderko
authors.forename: Audrey B
authors.initials: AB
authors.lastname: Bergeron
authors.forename: Andrew E
authors.initials: AE
authors.lastname: Christie
authors.forename: Patsy S
authors.initials: PS
authors.lastname: Dickinson
mesh_terms: D000818:Animals; D016477:Artifacts; D002853:Chromatography, Liquid; D003080:Cold Temperature; D006358:Hot Temperature; D059625:Liquid-Liquid Extraction; D000432:Methanol / Q000737:chemistry*; D008745:Methylation; D008121:Nephropidae; D009479:Neuropeptides / Q000737:chemistry* / Q000378:metabolism*; D011480:Protease Inhibitors / Q000737:chemistry / Q000378:metabolism; D059748:Proteolysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D009479:Neuropeptides; D011480:Protease Inhibitors; C079067:orcokinin; D000432:Methanol
keywords: CoG; Crustacean neuropeptides; HPLC Chip–nanoESI Q-TOF MS; LG; MALDI-FTMS; ME; MI; MT; Matrix assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS); Methanol; Nanoelectrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI Q-TOF MS); Neuropeptide extraction; Orcokinin; PO; SG; SORI-CID; STG; SoG/brain; X-organ; XO; commissural ganglion; high performance liquid chromatography-Chip nano-electrospray ionization quadrupole time-of-flight mass spectrometry; lamina ganglionaris; matrix assisted laser desorption/ionization Fourier transform mass spectrometry; medulla externa; medulla interna; medulla terminalis; pericardial organ; sinus gland; stomatogastric ganglion; supraesophageal ganglion; sustained off-resonance irradiation collision-induced dissociation