Programmable One-Tube Sialic Acid Derivatization Enables Dual-Mode N-Glycomics of Low-Input Samples.

Low-input glycomics remains particularly challenging for sialylated N-glycans because rigorous linkage-specific derivatization often increases sample handling, disperses signal across multiple linkage isomers, and compromises sensitivity. Here, we report a programmable one-tube workflow that integrates cell lysis, N-glycan release, sialic acid derivatization, and purification in a single microcentrifuge tube for highly sensitive N-glycome profiling. By controlling ammonia activity and reaction time after ethyl esterification, the workflow enables optional sialic acid linkage-specific, linkage-nonspecific, and sequential dual-mode analyses on the same low-input sample. On a standard mass spectrometry platform, the method demonstrated direct N-glycomic profiling from nanogram-scale glycoprotein inputs, nanoliter-scale human serum, and only a few thousand primary T cells. Applied to splenic B cells from an LPS-induced inflammatory model, a marked remodeling of the N-glycome from high-mannose species toward sialylated complex glycans was revealed, while maintaining an unchanged α2,3/α2,6 overall ratio. These results establish a chemistry-programmable route to dual-mode N-glycomics for low-input samples and expand the practical scope of linkage-resolved glycomic analysis in mass-limited biospecimens.