Extending native mass spectrometry approaches to integral membrane proteins.

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.

Sizing up DNA nanostructure assembly with native mass spectrometry and ion mobility.

Recent interest in biological and synthetic DNA nanostructures has highlighted the need for methods to comprehensively characterize intermediates and end products of multimeric DNA assembly. Here we use native mass spectrometry in combination with ion mobility to determine the mass, charge state and collision cross section of noncovalent DNA assemblies, and thereby elucidate their structural composition, oligomeric state, overall size and shape. We showcase the approach with a prototypical six-subunit DNA nanostructure to reveal how its assembly is governed by the ionic strength of the buffer, as well as how the mass and mobility of heterogeneous species can be well resolved by careful tuning of instrumental parameters. We find that the assembly of the hexameric, barrel-shaped complex is guided by positive cooperativity, while previously undetected higher-order 12- and 18-mer assemblies are assigned to defined larger-diameter geometric structures. Guided by our insight, ion mobility-mass spectrometry is poised to make significant contributions to understanding the formation and structural diversity of natural and synthetic oligonucleotide assemblies relevant in science and technology.

Interrogating Membrane Protein Structure and Lipid Interactions by Native Mass Spectrometry.

Native mass spectrometry and native ion mobility mass spectrometry are now established techniques in structural biology, with recent work developing these methods for the study of integral membrane proteins reconstituted in both lipid bilayer and detergent environments. Here we show how native mass spectrometry can be used to interrogate integral membrane proteins, providing insights into conformation, oligomerization, subunit composition/stoichiometry, and interactions with detergents/lipids/drugs. Furthermore, we discuss the sample requirements and experimental considerations unique to integral membrane protein native mass spectrometry research.

Native Mass Spectrometry for the Characterization of Structure and Interactions of Membrane Proteins.

Over the past years, native mass spectrometry and ion mobility have grown into techniques that are widely applicable to the study of aspects of protein structure. More recently, it has become apparent that this approach provides a very promising avenue for the investigation of integral membrane proteins in lipid or detergent environments.In this chapter, we discuss applications of native mass spectrometry and ion mobility in membrane protein research-what is important to take into consideration when working with membrane proteins, and what the requirements are for sample preparation for native mass spectrometry. Furthermore, we will discuss the types of information provided by the measurements, including the oligomeric state, subunit composition and stoichiometry, interactions with detergents or lipids, conformational transitions, and the binding and structural effect of ligands and drugs.