title: Characterization of Membrane Protein-Lipid Interactions by Mass Spectrometry Ion Mobility Mass Spectrometry.
abstract: Lipids in the biological membrane can modulate the structure and function of integral and peripheral membrane proteins. Distinguishing individual lipids that bind selectively to membrane protein complexes from an ensemble of lipid-bound species remains a daunting task. Recently, ion mobility mass spectrometry (IM-MS) has proven to be invaluable for interrogating the interactions between protein and individual lipids, where the complex undergoes collision induced unfolding followed by quantification of the unfolding pathway to assess the effect of these interactions. However, gas-phase unfolding experiments for membrane proteins are typically performed on the entire ensemble (apo and lipid bound species), raising uncertainty to the contribution of individual lipids and the species that are ejected in the unfolding process. Here, we describe the application of mass spectrometry ion mobility mass spectrometry (MS-IM-MS) for isolating ions corresponding to lipid-bound states of a model integral membrane protein, ammonia channel (AmtB) from Escherichia coli. Free of ensemble effects, MS-IM-MS reveals that bound lipids are ejected as neutral species; however, no correlation was found between the lipid-induced stabilization of complex and their equilibrium binding constants. In comparison to data obtained by IM-MS, there are surprisingly limited differences in stability measurements from IM-MS and MS-IM-MS. The approach described here to isolate ions of membrane protein complexes will be useful for other MS methods, such as surface induced dissociation or collision induced dissociation to determine the stoichiometry of hetero-oligomeric membrane protein complexes. Graphical Abstract ᅟ.
authors.affiliation: Center for Infectious and Inflammatory Diseases, Institute of Biosciences and Technology, Texas A&M University, Houston, TX, 77030, USA.
authors.forename: Yang
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Center for Infectious and Inflammatory Diseases, Institute of Biosciences and Technology, Texas A&M University, Houston, TX, 77030, USA.
authors.forename: Xiao
authors.initials: X
authors.lastname: Cong
authors.affiliation: Center for Infectious and Inflammatory Diseases, Institute of Biosciences and Technology, Texas A&M University, Houston, TX, 77030, USA.
authors.forename: Wen
authors.initials: W
authors.lastname: Liu
authors.affiliation: Center for Infectious and Inflammatory Diseases, Institute of Biosciences and Technology, Texas A&M University, Houston, TX, 77030, USA. ALaganowsky@ibt.tamhsc.edu.|Department of Chemistry, Texas A&M University, College Station, TX, 77842, USA. ALaganowsky@ibt.tamhsc.edu.|Department of Microbial Pathogenesis and Immunology, College of Medicine, Texas A&M University, Bryan, TX, 77807, USA. ALaganowsky@ibt.tamhsc.edu.
authors.forename: Arthur
authors.identifier: 0000-0001-5012-5547
authors.initials: A
authors.lastname: Laganowsky
mesh_terms: D027682:Cation Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D000075663:Ion Mobility Spectrometry / Q000379:methods*; D007477:Ions / Q000737:chemistry / Q000378:metabolism; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D055550:Protein Stability; D058767:Protein Unfolding; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: C111096:AmtB protein, E coli; D027682:Cation Transport Proteins; D029968:Escherichia coli Proteins; D007477:Ions; D008563:Membrane Lipids
keywords: Ammonium channel; Collision induced unfolding; Lipids; Mass spectrometry of intact membrane protein complexes; Membrane protein lipid interactions; Membrane proteins; Native mass spectrometry