Mass Spectrometry-Based Shotgun Glycomics Using Labeled Glycan Libraries.

Area of Science:

• Glycomics
• Mass Spectrometry
• Biochemistry

Background:

• Mass spectrometry-based shotgun glycomics (MS-SG) is a sensitive method for discovering natural ligands of glycan-binding proteins (GBPs).
• Determining glycan concentrations is difficult, preventing direct measurement of ligand affinities.
• Relative affinities can be estimated by combining MS-SG data with hydrophilic interaction liquid chromatography (HILIC) for relative concentration determination.

Purpose of the Study:

• To validate the MS-SG approach for ranking glycan ligand affinities.
• To assess the feasibility of performing catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) directly on labeled glycans.
• To compare different fluorophore labels for their impact on affinity rankings.

Main Methods:

• Libraries of labeled N-glycans from human cells and tissues were prepared using 2-aminobenzamide (2-AB), 2-aminobenzoic acid (2-AA), or procainamide (proA).
• Labeled glycan libraries were screened against plant and human GBPs using CaR-ESI-MS.
• Glycan concentrations were quantified using HILIC.

Main Results:

• Significant differences in affinity rankings were observed with different fluorophore labels and labeling approaches.
• Labeling efficiencies, label-induced affinity changes, and CaR-ESI-MS release efficiencies influenced rankings.
• The 2-AB labeling combined with CaR-ESI-MS and HILIC provided the most consistent results.

Conclusions:

• The choice of fluorophore label significantly impacts glycan-binding protein (GBP) affinity rankings in MS-SG.
• The 2-AB labeling method is recommended for MS-SG due to its reliability in describing GBP binding specificities for N-glycans.
• This study highlights the importance of careful method selection for accurate glycomics analysis.