Chromatographic Phospholipid Trapping for Automated H/D Exchange Mass Spectrometry of Membrane Protein–Lipid Assemblies

Dietmar Hammerschmid; Valeria Calvaresi; Chloe Bailey; Ben Russell Lewis; Argyris Politis; Michael Morris; Laetitia Denbigh; Malcolm Anderson; Eamonn Reading

doi:10.1021/acs.analchem.2c04876

Chromatographic Phospholipid Trapping for Automated H/D Exchange Mass Spectrometry of Membrane Protein–Lipid Assemblies

Dietmar Hammerschmid^*, Valeria Calvaresi, Chloe Bailey, Ben Russell Lewis, Argyris Politis, Michael Morris, Laetitia Denbigh, Malcolm Anderson, Eamonn Reading^*

^*Corresponding author for this work

Chemistry

SYNLAB International GmbH

Research output: Contribution to journal › Article › peer-review

6 Citations (Scopus)

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.

Original language	English
Pages (from-to)	3002-3011
Number of pages	10
Journal	Analytical Chemistry
Volume	95
Issue number	5
Early online date	27 Jan 2023
DOIs	https://doi.org/10.1021/acs.analchem.2c04876
Publication status	Published - 7 Feb 2023

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title = "Chromatographic Phospholipid Trapping for Automated H/D Exchange Mass Spectrometry of Membrane Protein–Lipid Assemblies",

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.",

author = "Dietmar Hammerschmid and Valeria Calvaresi and Chloe Bailey and {Russell Lewis}, Ben and Argyris Politis and Michael Morris and Laetitia Denbigh and Malcolm Anderson and Eamonn Reading",

note = "Funding Information: Work at King{\textquoteright}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{\textquoteright}s College London iCASE Studentship with Waters Corporation and B.R.L. by a King{\textquoteright}s College London Studentship. V.C. was supported by the Leverhulme Trust (RPG-2019-178) to A.P. and a King{\textquoteright}s College London funded research associate position to E.R. A.P. is an EPSRC Research Fellow (EP/V011715/1). Publisher Copyright: {\textcopyright} 2023 The Authors. Published by American Chemical Society.",

year = "2023",

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doi = "10.1021/acs.analchem.2c04876",

language = "English",

volume = "95",

pages = "3002--3011",

journal = "Analytical Chemistry",

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T1 - Chromatographic Phospholipid Trapping for Automated H/D Exchange Mass Spectrometry of Membrane Protein–Lipid Assemblies

AU - Hammerschmid, Dietmar

AU - Calvaresi, Valeria

AU - Bailey, Chloe

AU - Russell Lewis, Ben

AU - Politis, Argyris

AU - Morris, Michael

AU - Denbigh, Laetitia

AU - Anderson, Malcolm

AU - Reading, Eamonn

N1 - 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.

PY - 2023/2/7

Y1 - 2023/2/7

N2 - 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.

AB - 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.

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U2 - 10.1021/acs.analchem.2c04876

DO - 10.1021/acs.analchem.2c04876

M3 - Article

SN - 0003-2700

VL - 95

SP - 3002

EP - 3011

JO - Analytical Chemistry

JF - Analytical Chemistry

IS - 5

ER -

Chromatographic Phospholipid Trapping for Automated H/D Exchange Mass Spectrometry of Membrane Protein–Lipid Assemblies

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