Native mass spectrometry - a versatile tool for the study of challenging targets in structural biology
27 April 2016
Campus Groenenborger, V0.09 - Groenenborgerlaan 171 - 2020 Antwerpen
Organization / co-organization:
Department of Chemistry
Frank Sobott & Anne-Marie Lambeir
PhD defence of Mr. Albert Konijnenberg - Department of Chemistry
Mass spectrometry is the defining technique in proteome research, which aims at characterizing in detail key molecules such as proteins involved in biological processes in vitro. Developments of ‘soft’ or ‘nondenaturing’ ionization techniques have made it possible to transfer proteins and protein complexes intact into the gas-phase, thus making studies on the 3D structure of a protein amenable. In recent years, this field called “native mass spectrometry” has found its niche among the traditional structural techniques. Solving the 3D structure of a protein is important as such knowledge allows for the rational design of drugs. So far, the application of native mass spectrometry has been largely limited to the study of globular proteins and large non-covalent protein complexes. Although mass spectrometry is very successful in studying such systems, they are often also accessible for studying by traditional biophysical techniques, yielding structural information at much higher resolution. In this thesis we describe how native mass spectrometry can also be applied to study integral membrane proteins and intrinsically disordered proteins. Each of these protein classes poses a formidable challenge for studying by traditional biophysical techniques: integral membrane proteins are only stable in a lipid bilayer and isolation requires solubilizaton by detergents - compounds that are often incompatible with other biophysical techniques - whilst intrinsically flexible proteins display little to no secondary structure elements and conformational promiscuity, whereas a homogeneous sample is required for most biophysical techniques. We show that the challenges that intrinsically disordered proteins and membrane proteins pose can be matched by a set of state-of-the-art mass spectrometric methods that we developed. We prove the applicability of these methods by studying the gating of an ion channel in the gas phase, identify membrane embedded regions of integral membrane proteins, map the surfaces of large non-covalent complexes and even provide the gas phase structure of an intrinsically disordered protein. The obtained mass spectrometric results were further validated by other biophysical techniques or computational modelling and show how different approaches yield different pieces of structural information, each with their own benefit towards studying the 3D structure of proteins in the gas phase. Our results give a tantalizing view on the levels of structural information that native mass spectrometry can deliver.