N-Terminal Coumarin Derivatization-Aided De Novo Peptide Sequencing and Its Application to Peptidomics Using...

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 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 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.