N-Terminal Coumarin Derivatization-Aided De Novo Peptide Sequencing and Its Application to Peptidomics Using LC-Trapped Ion Mobility Spectrometry-qTOF/MS
peer-reviewed · Analytical Chemistry · 2026
| Date | 2026-06-30 |
| Type | peer-reviewed |
| Venue | Analytical Chemistry |
| Publisher | American Chemical Society (ACS) |
| Contribution | adjacent |
| DOI | 10.1021/acs.analchem.6c01542 |
| Venue 2-year citedness | 6.29 |
Preprint version: N-terminal Coumarin Derivatization-aided De Novo Peptide Sequencing and its Application to Peptidomics using LC-trapped Ion Mobility Spectrometry-qTOF/MS (2026-03-03, ChemRxiv)
Abstract
De novo peptide sequencing using LC-MS/MS enables database-independent sequence determination and facilitates the discovery of bioactive peptides and biomarkers in untargeted peptidomics. However, conventional MS/MS analyses often produce 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 b 1 -b 3 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 b 1 -b 9 ions. Notably, intact de novo sequencing identified 74 peptides with 32 misidentified 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 misidentification, 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