TY - JOUR
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.
UR - http://www.scopus.com/inward/record.url?scp=85147098986&partnerID=8YFLogxK
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 -