Extending native mass spectrometry approaches to integral membrane proteins

Abstract

Recent developments in native mass spectrometry and ion mobility have made it possible to analyze the composition and structure of membrane protein complexes in the gas-phase. In this short review we discuss the experimental strategies that allow to elucidate aspects of the dynamic structure of these important drug targets, such as the structural effects of lipid binding or detection of co-populated conformational and assembly states during gating on an ion channel. As native mass spectrometry relies on nano-electrospray of natively reconstituted proteins, a number of commonly used lipid- and detergent-based reconstitution systems have been evaluated for their compatibility with this approach, and parameters for the release of intact, native-like folded membrane proteins studied in the gas-phase. The strategy thus developed can be employed for the investigation of the subunit composition and stoichiometry, oligomeric state, conformational changes, and lipid and drug binding of integral membrane proteins.

Lipids Shape the Electron Acceptor-Binding Site of the Peripheral Membrane Protein Dihydroorotate Dehydrogenase

“…Yet the highly amphipathic nature of these proteins and their dependence on lipid interactions continue to limit structural investigations. It has recently been established that non-denaturing nano-electrospray ionization mass spectrometry (nESI-MS) is well suited to study integral membrane protein complexes ( Konijnenberg et al., 2015 , Landreh and Robinson, 2015 ). Here, the protein is ionized while embedded in a protective detergent micelle that is subsequently removed by collisional activation to release the intact complex for MS analysis ( Barrera et al., 2008 ).…”

193 nm Ultraviolet Photodissociation Mass Spectrometry of Tetrameric Protein Complexes Provides Insight into Quaternary and Secondary Protein Topology

“…2 The development of electrospray ionization and particularly native electrospray ionization has permitted transfer of multiprotein complexes into the gas phase and has consequently transformed the ability to examine protein complexes by mass spectrometry. 3–6 Characterization of the assembly and architecture of multiprotein complexes via their solution and gas-phase disassembly pathways has gained momentum as a viable approach in structural biology. 7–9 Dissociation of protein complexes in a manner that reflects the organization of the multi-protein complex, however, has proved challenging.…”

Characterization of hydrogen bonding motifs in proteins: hydrogen elimination monitoring by ultraviolet photodissociation mass spectrometry

“…1,2 In native mass spectrometry, intact, folded proteins and protein complexes are ionized from native buffers, providing a means to study the native-like species in the gas phase and affording direct stoichiometric and dynamics information. Native mass spectrometry has proven valuable in the development of conditions for crystallization of membrane proteins 3,4 and for exploration of the conformational space occupied by intrinsically disordered proteins, 5,6 both of which are classes of proteins that are difficult to characterize by conventional structural biology methods. Despite the success of these applications, however, the structural information obtainable from mass spectrometry is inherently low resolution and frequently only sheds light on gross structural features.…”