Elusive Protein-Glycosphingolipid Interactions Revealed by Membrane Anchor-Assisted Native Mass Spectrometry

Area of Science:

• Biochemistry
• Glycobiology
• Analytical Chemistry

Background:

• Glycan-binding proteins (GBPs) interact with glycosphingolipids (GSLs) on cell membranes, influencing biological processes.
• Studying these interactions is difficult due to limited purified GSLs and weak binding affinities.
• Native mass spectrometry (nMS) shows promise for identifying GBP ligands but struggles with low-affinity interactions.

Purpose of the Study:

• To develop a novel method, MEAN-nMS, for detecting low-affinity GBP-GSL complexes.
• To improve the identification of GSL ligands for GBPs.
• To enable GSL ligand detection directly from intact cells.

Main Methods:

• Membrane Anchor-assisted native Mass Spectrometry (MEAN-nMS) was developed, using a membrane anchor to localize GBPs and enhance GSL binding.
• A catch-and-release (CaR) strategy (MEAN-CaR-nMS) was employed for GSL detection after release from GBP-GSL complexes.
• Purified gangliosides in nanodiscs were screened against human immune lectins, and results were compared to standard nMS.

Main Results:

• MEAN-nMS successfully detected low-affinity GBP-GSL complexes, overcoming limitations of standard nMS which yielded false negatives.
• All tested ligands were identified by MEAN-(CaR)-nMS without false positives.
• The method was validated by screening natural GSL libraries against human and viral proteins, and GSL ligands were detected directly from cells.

Conclusions:

• MEAN-nMS is a reliable and sensitive method for discovering GSL ligands, especially for weak interactions.
• The technique significantly advances the ability to study GBP-GSL interactions.
• MEAN-CaR-nMS enables shotgun glycomics screening using intact cells, opening new avenues in biological research.