N-terminal Coumarin Derivatization-aided De Novo Peptide Sequencing and its Application to Peptidomics using LC-trapped Ion Mobility Spectrometry-qTOF/MS
preprint · ChemRxiv · 2026
| Date | 2026-03-03 |
| Type | preprint |
| Venue | ChemRxiv |
| Publisher | ChemRxiv |
| Contribution | adjacent |
| DOI | 10.26434/chemrxiv.15000573/v1 |
Peer-reviewed version: N-Terminal Coumarin Derivatization-Aided De Novo Peptide Sequencing and Its Application to Peptidomics Using LC-Trapped Ion Mobility Spectrometry-qTOF/MS (2026-06-30, Analytical Chemistry)
Abstract
De novo peptide sequencing using LC-MS/MS enables database-independent sequence determination and facilitates the discovery of novel bioactive peptides and biomarkers in untargeted peptidomics. However, conventional MS/MS analyses often produces fragment ions that are insufficient for complete sequencing. Here, we established an LC-MS/MS workflow using N -succinimidyl 7-methoxycoumarin-3-carboxylate (Me-Cou) as an N -terminal tag to improve de novo sequencing performance. Me-Cou derivatization generated highly informative fragment ions, particularly b₁-b₃ ions, for (Gly)₄ at 1 μmol/L, surpassing the performance of established tags, such as 2,4,6-trinitrobenzenesulfonic acid, 3-aminopyridyl-hydroxy-succinimidyl carbamate, and N -succinimidyl [tris(2,4,6-trimethoxyphenyl)phosphonio]acetate bromide. The Me-Cou tag also enabled confident sequencing of (Gly)₁₀, generating abundant b₁-b9 ions. Notably, intact de novo sequencing identified 74 peptides with 32 misannotated peptides that were not included in a 132 standard peptide mixture (86 dipeptides and 46 oligopeptides). However, all peptides were correctly identified using Me-Cou-aided de novo sequencing with no misannotations, demonstrating high specificity and accuracy. Additionally, Me-Cou-aided de novo sequencing successfully identified 328 peptides in casein peptone, all of which were assigned as casein protein fragments. Overall, Me-Cou-based LC-MS/MS significantly enhanced peptide detection and the characterization of sequence diversity compared with intact analysis. This methodology is a promising strategy for untargeted peptidomics, enabling comprehensive characterization of endogenous peptides in both protein hydrolysates and biological fluids.
Methods and tools
- Me-Cou N-terminal derivatization de novo sequencing: N-terminal derivatization with N-succinimidyl 7-methoxycoumarin-3-carboxylate (Me-Cou), which boosts low-mass b-ion yield so de novo sequencing can read the N-terminal residues, applied to untargeted peptidomics on LC-TIMS-qTOF/MS.
- PEAKS: Commercial DP-based de novo