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.