C-terminal methylation of truncated neuropeptides: An enzyme- assistedextraction artifact involving methanol
Neuropeptides are the largest class of signaling molecules used by nervous systems. Today, neuropeptidediscovery commonly involves chemical extraction from a tissue source followed by mass spectrometriccharacterization. Ideally, the extraction procedure accurately preserves the sequence and any inher-ent 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 fam-ily members, NFDEIDRSGFG-OMe and SSEDMDRLGFG-OMe, are non-native peptides generated fromfull-length orcokinin precursors as the result of a highly selective peptide modification (peptide trun-cation with C-terminal methylation) that occurs during extraction. These peptides were observed byMALDI-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 methanolexcluded 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 substitutingdeuterated 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 isnot produced via a chemical acid-catalyzed esterification. Instead, the methylated peptide appears toresult from proteolytic truncation in the presence of methanol, as evidenced by a reduction in conver-sion with the addition of a protease-inhibitor cocktail; heat effectively eliminated the conversion. Thisunusual and highly specific extraction-derived peptide conversion exemplifies the need to consider bothchemical and biochemical processes that may modify the structure of endogenous neuropeptides. © 2013 The Authors. Published by Elsevier Inc. All rights reserved.
