Mass spectrometry-enabled structural biology of membrane proteins

Abstract

a b s t r a c tThe last $25 years has seen mass spectrometry (MS) emerge as an integral method in the structural biology toolkit. In particular, MS has enabled the structural characterization of proteins and protein assemblies that have been intractable by other methods, especially those that are large, heterogeneous or transient, providing experimental evidence for their structural organization in support of, and in advance of, high resolution methods. The most recent frontier conquered in the field of MS-based structural biology has been the application of established methods for studying water soluble proteins to the more challenging targets of integral membrane proteins. The power of MS in obtaining structural information has been enabled by advances in instrumentation and the development of hyphenated mass spectrometry-based methods, such as ion mobility spectrometry-MS, chemical crosslinking-MS and other chemical labelling/footprinting-MS methods. In this review we detail the insights garnered into the structural biology of membrane proteins by applying such techniques. Application and refinement of these methods has yielded unprecedented insights in many areas, including membrane protein conformation, dynamics, lipid/ligand binding, and conformational perturbations due to ligand binding, which can be challenging to study using other methods.

Fast photochemical oxidation of proteins (FPOP): A powerful mass spectrometry–based structural proteomics tool

“…Until recently, X-ray crystallography, HDX, and NMR among others have been used by structural biologists to make seminal contributions (82). Where those techniques were lacking, cryo-electron microscopy (cryo-EM) picked up the slack to gain structural information of MPs (83).…”

Importance of collision cross section measurements by ion mobility mass spectrometry in structural biology

“…However, they contribute less than 1% of the structures currently deposited in the PDB. Relatively recent developments in native MS have seen renewed interest in the analysis of membrane proteins by this method, with the realization that membrane proteins can be transferred to the gas phase while encapsulated in lipid or detergent environments, before being thermally activated to strip away the solubilizing lipid/detergent environment and enable mass or IM separation . While the influence of this activating stage must be carefully considered and controlled in order to minimize structural perturbations that would contribute to flawed CCS measurement, recent examples highlight the exciting possibilities in utilizing IM‐MS for structural characterization of membrane protein complexes and the structural changes associated with lipid and other small molecule interactions …”

“…B, Homology modelling allows an atomic-level structure of the protein subunit to be built, along with higher order structures such as the tail-to-tail dimer conformation and ring-like arrangement of the hexamer, satisfying IM restraints. Reproduced from Politis et al58 under the conditions of the creative commons attribution license thermally activated to strip away the solubilizing lipid/detergent environment and enable mass or IM separation 60. While the influence of this activating stage must be carefully considered and controlled in order to minimize structural perturbations that would contributeto flawed CCS measurement, recent examples highlight the exciting possibilities in utilizing IM-MS for structural characterization of membrane protein complexes and the structural changes associated with lipid and other small molecule interactions.…”

Characterizing the lipid-protein interface of the human serotonin transporter by crosslinking mass spectrometry

“…Cholesterol photoaffinity probes have also characterized lipid-protein interactions with ion channels (27,28). The advent of mass spectrometry (MS) methodologies offers the opportunity to examine membrane protein structure (29) to more sensitively and accurately detect and map the interaction of lipids with membrane proteins (30)(31)(32). In this study, we conduct studies analogous to those used in examining cholesterol interactions with the glycine receptor (33) to directly probe the lipid accessible regions of hSERT in its apo-state.…”