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Cross-Linking Mass Spectrometry on P-Glycoprotein

The ABC transporter P-glycoprotein (Pgp) has been found to be involved in multidrug resistance in tumor cells. Lipids and cholesterol have a pivotal role in Pgp’s conformations; however, it is often difficult to investigate it with conventional structural biology techniques. Here, we applied robust...

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Autores principales: Gellen, Gabriella, Klement, Eva, Biwott, Kipchumba, Schlosser, Gitta, Kalló, Gergő, Csősz, Éva, Medzihradszky, Katalin F., Bacso, Zsolt
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10341432/
https://www.ncbi.nlm.nih.gov/pubmed/37445813
http://dx.doi.org/10.3390/ijms241310627
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author Gellen, Gabriella
Klement, Eva
Biwott, Kipchumba
Schlosser, Gitta
Kalló, Gergő
Csősz, Éva
Medzihradszky, Katalin F.
Bacso, Zsolt
author_facet Gellen, Gabriella
Klement, Eva
Biwott, Kipchumba
Schlosser, Gitta
Kalló, Gergő
Csősz, Éva
Medzihradszky, Katalin F.
Bacso, Zsolt
author_sort Gellen, Gabriella
collection PubMed
description The ABC transporter P-glycoprotein (Pgp) has been found to be involved in multidrug resistance in tumor cells. Lipids and cholesterol have a pivotal role in Pgp’s conformations; however, it is often difficult to investigate it with conventional structural biology techniques. Here, we applied robust approaches coupled with cross-linking mass spectrometry (XL-MS), where the natural lipid environment remains quasi-intact. Two experimental approaches were carried out using different cross-linkers (i) on living cells, followed by membrane preparation and immunoprecipitation enrichment of Pgp, and (ii) on-bead, subsequent to membrane preparation and immunoprecipitation. Pgp-containing complexes were enriched employing extracellular monoclonal anti-Pgp antibodies on magnetic beads, followed by on-bead enzymatic digestion. The LC-MS/MS results revealed mono-links on Pgp’s solvent-accessible residues, while intraprotein cross-links confirmed a complex interplay between extracellular, transmembrane, and intracellular segments of the protein, of which several have been reported to be connected to cholesterol. Harnessing the MS results and those of molecular docking, we suggest an epitope for the 15D3 cholesterol-dependent mouse monoclonal antibody. Additionally, enriched neighbors of Pgp prove the strong connection of Pgp to the cytoskeleton and other cholesterol-regulated proteins. These findings suggest that XL-MS may be utilized for protein structure and network analyses in such convoluted systems as membrane proteins.
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spelling pubmed-103414322023-07-14 Cross-Linking Mass Spectrometry on P-Glycoprotein Gellen, Gabriella Klement, Eva Biwott, Kipchumba Schlosser, Gitta Kalló, Gergő Csősz, Éva Medzihradszky, Katalin F. Bacso, Zsolt Int J Mol Sci Article The ABC transporter P-glycoprotein (Pgp) has been found to be involved in multidrug resistance in tumor cells. Lipids and cholesterol have a pivotal role in Pgp’s conformations; however, it is often difficult to investigate it with conventional structural biology techniques. Here, we applied robust approaches coupled with cross-linking mass spectrometry (XL-MS), where the natural lipid environment remains quasi-intact. Two experimental approaches were carried out using different cross-linkers (i) on living cells, followed by membrane preparation and immunoprecipitation enrichment of Pgp, and (ii) on-bead, subsequent to membrane preparation and immunoprecipitation. Pgp-containing complexes were enriched employing extracellular monoclonal anti-Pgp antibodies on magnetic beads, followed by on-bead enzymatic digestion. The LC-MS/MS results revealed mono-links on Pgp’s solvent-accessible residues, while intraprotein cross-links confirmed a complex interplay between extracellular, transmembrane, and intracellular segments of the protein, of which several have been reported to be connected to cholesterol. Harnessing the MS results and those of molecular docking, we suggest an epitope for the 15D3 cholesterol-dependent mouse monoclonal antibody. Additionally, enriched neighbors of Pgp prove the strong connection of Pgp to the cytoskeleton and other cholesterol-regulated proteins. These findings suggest that XL-MS may be utilized for protein structure and network analyses in such convoluted systems as membrane proteins. MDPI 2023-06-25 /pmc/articles/PMC10341432/ /pubmed/37445813 http://dx.doi.org/10.3390/ijms241310627 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Gellen, Gabriella
Klement, Eva
Biwott, Kipchumba
Schlosser, Gitta
Kalló, Gergő
Csősz, Éva
Medzihradszky, Katalin F.
Bacso, Zsolt
Cross-Linking Mass Spectrometry on P-Glycoprotein
title Cross-Linking Mass Spectrometry on P-Glycoprotein
title_full Cross-Linking Mass Spectrometry on P-Glycoprotein
title_fullStr Cross-Linking Mass Spectrometry on P-Glycoprotein
title_full_unstemmed Cross-Linking Mass Spectrometry on P-Glycoprotein
title_short Cross-Linking Mass Spectrometry on P-Glycoprotein
title_sort cross-linking mass spectrometry on p-glycoprotein
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10341432/
https://www.ncbi.nlm.nih.gov/pubmed/37445813
http://dx.doi.org/10.3390/ijms241310627
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