Chromatographic phospholipid trapping for automated H/D exchange mass spectrometry of membrane protein-lipid assemblies
Chromatographic phospholipid trapping for automated H/D exchange mass spectrometry of membrane protein-lipid assemblies
Lipid interactions modulate the function, folding, structure, and organization of membrane proteins. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has emerged as a useful tool to understand the structural dynamics of these proteins within lipid environments. Lipids, however, have proven problematic for HDX-MS analysis of membrane-embedded proteins due to their presence of impairing proteolytic digestion, causing liquid chromatography column fouling, ion suppression, and/or mass spectral overlap. Herein, we describe the integration of a chromatographic phospholipid trap column into the HDX-MS apparatus to enable online sample delipidation prior to protease digestion of deuterium-labeled protein-lipid assemblies. We demonstrate the utility of this method on membrane scaffold protein-lipid nanodisc─both empty and loaded with the ∼115 kDa transmembrane protein AcrB─proving efficient and automated phospholipid capture with minimal D-to-H back-exchange, peptide carry-over, and protein loss. Our results provide insights into the efficiency of phospholipid capture by ZrO2-coated and TiO2 beads and describe how solution conditions can be optimized to maximize not only the performance of our online but also the existing offline, delipidation workflows for HDX-MS. We envision that this HDX-MS method will significantly ease membrane protein analysis, allowing to better interrogate their dynamics in artificial lipid bilayers or even native cell membranes.
3002-3011
Hammerschmid, Dietmar
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Calvaresi, Valeria
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Bailey, Chloe
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Russell Lewis, Benjamin
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Politis, Argyris
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Morris, Michael
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Denbigh, Laetitia
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Anderson, Malcolm
57515f5d-235e-42bc-924c-93bf13605bb0
Reading, Eamonn
62fed933-f867-4c72-89e7-83aea573a836
7 February 2023
Hammerschmid, Dietmar
5934e033-df57-4533-8e58-645ed0315759
Calvaresi, Valeria
3c061892-a4da-428d-98e3-d277d406aec3
Bailey, Chloe
f0a3719f-84d8-4d2d-b095-53a77c815428
Russell Lewis, Benjamin
6052ce96-6af8-4c50-a49c-594782e76493
Politis, Argyris
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Morris, Michael
16a47b8d-2e37-41bf-ac7f-82baa9a057f3
Denbigh, Laetitia
cce71fdc-430d-4596-b934-65e5fd8e5bb6
Anderson, Malcolm
57515f5d-235e-42bc-924c-93bf13605bb0
Reading, Eamonn
62fed933-f867-4c72-89e7-83aea573a836
Hammerschmid, Dietmar, Calvaresi, Valeria, Bailey, Chloe, Russell Lewis, Benjamin, Politis, Argyris, Morris, Michael, Denbigh, Laetitia, Anderson, Malcolm and Reading, Eamonn
(2023)
Chromatographic phospholipid trapping for automated H/D exchange mass spectrometry of membrane protein-lipid assemblies.
Analytical Chemistry, 95 (5), .
(doi:10.1021/acs.analchem.2c04876).
Abstract
Lipid interactions modulate the function, folding, structure, and organization of membrane proteins. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has emerged as a useful tool to understand the structural dynamics of these proteins within lipid environments. Lipids, however, have proven problematic for HDX-MS analysis of membrane-embedded proteins due to their presence of impairing proteolytic digestion, causing liquid chromatography column fouling, ion suppression, and/or mass spectral overlap. Herein, we describe the integration of a chromatographic phospholipid trap column into the HDX-MS apparatus to enable online sample delipidation prior to protease digestion of deuterium-labeled protein-lipid assemblies. We demonstrate the utility of this method on membrane scaffold protein-lipid nanodisc─both empty and loaded with the ∼115 kDa transmembrane protein AcrB─proving efficient and automated phospholipid capture with minimal D-to-H back-exchange, peptide carry-over, and protein loss. Our results provide insights into the efficiency of phospholipid capture by ZrO2-coated and TiO2 beads and describe how solution conditions can be optimized to maximize not only the performance of our online but also the existing offline, delipidation workflows for HDX-MS. We envision that this HDX-MS method will significantly ease membrane protein analysis, allowing to better interrogate their dynamics in artificial lipid bilayers or even native cell membranes.
Text
acs.analchem.2c04876
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More information
Accepted/In Press date: 13 January 2023
e-pub ahead of print date: 27 January 2023
Published date: 7 February 2023
Additional Information:
Funding Information:
Work at King’s College London by D.H. and E.R. was supported by a UKRI Future Leader Fellowship (MR/S015426/1) to E.R. C.B. was supported by a King’s College London iCASE Studentship with Waters Corporation and B.R.L. by a King’s College London Studentship. V.C. was supported by the Leverhulme Trust (RPG-2019-178) to A.P. and a King’s College London funded research associate position to E.R. A.P. is an EPSRC Research Fellow (EP/V011715/1).
Publisher Copyright:
© 2023 The Authors. Published by American Chemical Society.
Identifiers
Local EPrints ID: 478873
URI: http://eprints.soton.ac.uk/id/eprint/478873
ISSN: 0003-2700
PURE UUID: a023b7a6-e295-4ac7-aab2-b512183031a1
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Date deposited: 12 Jul 2023 16:34
Last modified: 17 Mar 2024 04:19
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Contributors
Author:
Dietmar Hammerschmid
Author:
Valeria Calvaresi
Author:
Chloe Bailey
Author:
Benjamin Russell Lewis
Author:
Argyris Politis
Author:
Michael Morris
Author:
Laetitia Denbigh
Author:
Malcolm Anderson
Author:
Eamonn Reading
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