Membrane protein structural insights from chemical labeling and mass spectrometry

Abstract

Membrane proteins play a central role in virtually all biological processes, and they represent important drug targets. Unfortunately, the application of traditional high-resolution methods such as X-ray crystallography and NMR spectroscopy to membrane proteins remains challenging. This article reviews alternative approaches that involve chemical labeling and mass spectrometry (MS) for gaining insights into membrane protein structure, function, and interactions. Hydrogen/deuterium exchange MS represents an interesting avenue for exploring biomolecular conformations and dynamics, but thus far this technique has not been widely adopted for membrane protein studies. The main focus of this article is on the use of labeling agents that introduce covalent modifications in solvent-accessible protein regions. While it is possible to monitor the occurrence of single-site modifications using traditional biochemical methods or optical spectroscopy, the use of MS greatly enhances the scope and potential of this approach because multiple tagging events can be detected in parallel. The traditional bottom-up workflow of these studies involves the digestion of a chemically labeled membrane protein by a specific protease such as trypsin. This is followed by chromatographic separation of the resulting peptides and on-line electrospray ionization MS. The application of tandem MS allows pinpointing the exact locations of chemical modifications. A particularly exciting aspect is the applicability of covalent labeling techniques to membrane protein within their natural lipid environment, or even inside living cells. Some of these concepts will be illustrated using the oxidative labeling of bacteriorhodopsin as an example, but numerous other labeling agents and protein systems are being highlighted as well. It is hoped that this review will stimulate further developments in the characterization of membrane proteins.

Site-directed mutagenesis combined with oxidative methionine labeling for probing structural transitions of a membrane protein by mass spectrometry

“…The situation is particularly challenging for membrane proteins where digestion efficiencies under HDX conditions tend to be low [68], and where detergents often interfere with the analysis. It is therefore not surprising that membrane protein covalent labeling has become a fairly routine approach, whereas only a handful of HDX studies in this area have appeared over the past few years [28]. Luckily, however, several very recent studies indicate that rapid progress is now being made in the area of membrane protein HDX/MS [75][76][77].…”

“…Numerous MS-based covalent labeling experiments have been conducted on membrane proteins [28,29], e.g., for topologic investigations [30], as well as for monitoring conformational changes [29,[31][32][33][34] and binding interactions [35][36][37].…”

Hydrogen/deuterium exchange mass spectrometry and optical spectroscopy as complementary tools for studying the structure and dynamics of a membrane protein

“…The application of traditional high resolution structure determination methods such as X-ray crystallography [5] and NMR spectroscopy [6] to membrane proteins has been demonstrated, but the success rate of these strategies is low. Alternative techniques that are capable of providing low to medium level structural information are therefore of considerable interest [7,8].…”

Validation of Membrane Protein Topology Models by Oxidative Labeling and Mass Spectrometry

“…The location(s) and the extent of labeling can be determined by LC-MS/MS peptide mapping [31,32]. Such experiments have been used to explore conformational features and interactions of IMPs [32][33][34][35][36][37][38][39][40][41][42]. For IMP topology studies, however, this approach remains underutilized.…”