Cross-Linking Mass Spectrometry on P-Glycoprotein

Area of Science:

• Membrane protein structural biology
• Biochemistry
• Proteomics

Background:

• P-glycoprotein (Pgp), an ABC transporter, is implicated in multidrug resistance in tumor cells.
• Lipids and cholesterol significantly influence Pgp conformation, posing challenges for conventional structural studies.

Purpose of the Study:

• To investigate Pgp structure and interactions within its native lipid environment using cross-linking mass spectrometry (XL-MS).
• To explore the role of cholesterol in Pgp conformation and interactions.
• To identify Pgp-associated proteins and potential antibody epitopes.

Main Methods:

• Application of two XL-MS approaches on living cells and on-bead after membrane preparation and immunoprecipitation.
• Enrichment of Pgp-containing complexes using anti-Pgp antibodies and magnetic beads.
• LC-MS/MS analysis for identifying cross-linked peptides and protein neighbors.
• Molecular docking to suggest an antibody epitope.

Main Results:

• XL-MS identified solvent-accessible residues and confirmed interplay between Pgp segments, linking to cholesterol.
• An epitope for the 15D3 cholesterol-dependent antibody was proposed.
• Pgp was found to be strongly associated with the cytoskeleton and other cholesterol-regulated proteins.

Conclusions:

• XL-MS is a viable technique for analyzing membrane protein structure and networks in complex biological systems.
• This study provides insights into Pgp's structural organization and its interactions with lipids and cellular components.