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.

AMGSEFLamide, a member of a broadly conserved peptide family, modulates multiple neural networks inHomarus americanus

“…Individual brains were placed in 58 μl of LCMS water (Fisherbrand; Life Technologies Corp.) in a 1.5 ml low retention tube. The sample was heated at 100°C for 5 min to deactivate proteolytic enzymes (Stemmler et al, 2013) and was cooled to room temperature or stored at −20°C. For tissue extraction, methanol (CH 3 OH; LCMS-grade; Fisherbrand; 128 μl) and glacial acetic acid (CH 3 CO 2 H; reagent grade; >99%; Sigma-Aldrich, St Louis, MO, USA; 14 μl) were added to the tube.…”

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