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Automated removal of phospholipids from membrane proteins for H/D exchange mass spectrometry workflows



Kyle W. Anderson, Elyssia S. Gallagher, Jeffrey W. Hudgens


Membrane proteins are currently the most common targets for pharmaceuticals. However, characterization of their structural dynamics by hydrogen/deuterium exchange mass spectrometry (HDX- MS) is sparse due to insufficient automated methods to handle full-length membrane proteins in lipid bilayers. Additionally, membrane lipids used to mimic the membrane environment and to solubilize membrane proteins can impair chromatography performance and cause ion suppres-sion in the mass spectrometer. The workflow discussed herein advances HDX-MS capabilities and other MS applications for membrane proteins by providing a fully automated method for HDX-MS analysis based on a phospholipid removal scheme compatible with robotic handling. Phospholipids were depleted from protein samples by addition of zirconium oxide beads, which were subsequently removed by inline filtration using syringeless nanofilters. To demonstrate this method, single-pass transmembrane protein FcγRIIa (CD32a) expressed into liposomes was used. Successful depletion of phospholipids ensured optimal liquid chromatography mass spectrometry (LC-MS) performance, and measurement of peptides from the transmembrane domain of FcγRIIa indicated phospholipids associated with this region were either not present or did not shield the transmembrane domain from digestion by pepsin. Furthermore, amino acid sequence coverage provided by this method was suitable to enable future measurement of structural dynamics of ectodomain, transmembrane domain, and endodomain of FcγRIIa. Moreover, this method is the first to enable fully automated HDX-MS on full-length transmembrane proteins in lipid bilayers, a notable advancement to facilitate understanding of membrane proteins, development of pharmaceuticals, and characterization for regulatory agencies
Analytical Chemistry


Anderson, K. , Gallagher, E. and Hudgens, J. (2018), Automated removal of phospholipids from membrane proteins for H/D exchange mass spectrometry workflows, Analytical Chemistry, [online], (Accessed June 16, 2024)


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Created May 3, 2018, Updated November 10, 2018