Chromatographic phospholipid trapping for automated H/D exchange mass spectrometry analysis 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 impairing proteolytic digestion, causing liquid chromatography column fouling, ion suppression, and/or mass spectral overlap. Here, 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 with minimal 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 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 cell membranes.

Hydrogen/deuterium exchange-mass spectrometry of integral membrane proteins in native-like environments: current scenario and the way forward

“…Nonspecific protein adsorption onto the ZrO 2 column was also reported by Hammerschmid et al [62]. Whilst attempting to reduce nonspecific binding, less severe adsorption was observed over technical replicates, suggesting 'blocking' of nonspecific binding sites via protein saturation.…”

“…Hammerschmid et al proposed chromatographic phospholipid trapping columns containing ZrO 2 or titanium dioxide beads (TiO 2 ) [ 62 ]. In their method, a Waters HDX manager and UPLC was modified with an additional valve and ZrO 2 /TiO 2 column to perform on-line protein- or peptide-level delipidation.…”

“…When evaluating delipidation efficiency, Hammerschmid et al reported a >100 to 1000-fold reduction in POPC when using ZrO 2 /TiO 2 trap columns [ 62 ]. Furthermore, an approximately 96% delipidation efficiency was reported for E. coli lipid extract, demonstrating a capacity to handle complex lipid mixtures.…”

Structural mass spectrometry approaches to understand multidrug efflux systems

“…For HDX-MS, their lipid environment has long been a hurdle as lipids may hamper protein digestion and/or chromatographic peptide separation. However, recent advances have overcome this problem by allowing phospholipids to be removed predigestion using ZrO 2 beads [ 47 ], size-exclusion chromatography [ 48 ], or TCA precipitation [ 49 ], which will help to make membrane protein characterisation in HDX-MS more routine. XL and chemical labelling are relatively more tolerable towards membrane proteins as enough time is available for processing the permanently labelled protein for subsequent MS analysis.…”