Dual modifications strategy to quantify neutral and sialylated N-glycans simultaneously by MALDI-MS

Area of Science:

• Glycomics
• Mass Spectrometry
• Analytical Chemistry

Background:

• Direct quantitative comparison of neutral and sialylated glycans is hindered by differences in ionization efficiency during mass spectrometry.
• Existing methods lack the ability to simultaneously analyze and quantify both neutral and sialylated glycans accurately.

Purpose of the Study:

• To develop an integrated chemical strategy, Dual Reactions for Analytical Glycomics (DRAG), for the simultaneous quantitative comparison of neutral and sialylated glycans.
• To overcome the limitations of differing ionization efficiencies in mass spectrometric analysis of diverse glycan structures.

Main Methods:

• The DRAG strategy involves reductive amination of glycans with distinct isotope-labeled reagents followed by methylamidation of sialic acid residues.
• This process homogenizes ionization responses, allowing for direct comparison of neutral and sialylated glycans within a single mass spectrometry run.
• Utilized matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for signal detection and analysis.

Main Results:

• The DRAG strategy generates doublet signals with a static 6 Da mass difference, enabling quantitative assessment of glycan expression changes between samples.
• The method was validated using N-glycans from bovine fetuin and human serum IgG, demonstrating its chemical robustness.
• Subtle quantitative differences in urinary N-glycans were successfully measured when comparing morning and afternoon samples from a single donor.

Conclusions:

• The DRAG strategy provides a robust and accurate method for the simultaneous quantitative analysis of neutral and sialylated glycans.
• This approach significantly advances glycomics by enabling direct comparisons and facilitating the study of subtle glycan expression changes in biological samples.
• DRAG is applicable to various biological contexts, including the analysis of urinary N-glycans for biomarker discovery.