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

Area of Science:

• Glycomics
• Mass Spectrometry
• Biochemistry

Background:

• Analyzing sialylated N-glycans in low-input samples is challenging due to complex derivatization and sensitivity issues.
• Existing methods often require extensive sample handling and can lead to signal dispersion across linkage isomers.

Purpose of the Study:

• To develop a sensitive, programmable one-tube workflow for N-glycome profiling of low-input biological samples.
• To enable linkage-specific and dual-mode analysis of sialylated N-glycans using mass spectrometry.

Main Methods:

• Integration of cell lysis, N-glycan release, sialic acid derivatization, and purification in a single microcentrifuge tube.
• Control of ammonia activity and reaction time for optional linkage-specific, nonspecific, or sequential dual-mode sialic acid analyses.
• Application on a standard mass spectrometry platform for direct N-glycomic profiling.

Main Results:

• Demonstrated direct N-glycomic profiling from nanogram-scale glycoproteins, nanoliter-scale human serum, and thousands of primary T cells.
• Revealed significant N-glycome remodeling in splenic B cells from an inflammatory model, showing a shift towards sialylated complex glycans.
• Maintained an unchanged α2,3/α2,6 sialic acid linkage ratio during analysis.

Conclusions:

• Established a chemistry-programmable route for dual-mode N-glycomics, particularly beneficial for low-input samples.
• Expanded the scope of linkage-resolved glycomic analysis in mass-limited biospecimens.
• The workflow offers enhanced sensitivity and versatility for N-glycan profiling.