Painting proteins with covalent labels: What’s in the picture?

Abstract

Knowledge about the structural and biophysical properties of proteins when they are free in solution and/or in complexes with other molecules is essential for understanding the biological processes that proteins regulate. Such knowledge is also important to drug discovery efforts, particularly those focused on the development of therapeutic agents with protein targets. In the last decade a variety of different covalent labeling techniques have been used in combination with mass spectrometry to probe the solution-phase structures and biophysical properties of proteins and protein-ligand complexes. Highlighted here are five different mass spectrometry-based covalent labeling strategies including: continuous hydrogen/deuterium (H/D) exchange labeling, hydroxyl radical-mediated footprinting, SUPREX (stability of unpurified proteins from rates of H/D exchange), PLIMSTEX (protein-ligand interaction by mass spectrometry, titration, and H/D exchange), and SPROX (stability of proteins from rates of oxidation). The basic experimental protocols used in each of the above-cited methods are summarized along with the kind of biophysical information they generate. Also discussed are the relative strengths and weaknesses of the different methods for probing the wide range of conformational states that proteins and proteinligand complexes can adopt when they are in solution. (J Am Soc Mass Spectrom 2009, 20, 1193-1206) © 2009 Published by Elsevier Inc. on behalf of American Society for Mass Spectrometry P roteins fold into elaborate three-dimensional structures and, under native solution conditions, they spend a large fraction of time in highly compact structures. However, the solution-phase structures of proteins are not static. Proteins sample a wide range of conformational states in solution. The conformational changes that proteins undergo in solution can be as dramatic as a global unfolding event in which all higher-order structure is lost or as subtle as a breathing motion in which a specific element of secondary structure is partially unfolded in a more local unfolding event (see Figure 1). Knowledge about the structures, kinetics, and thermodynamics involved in the conformational changes that proteins undergo in solution and as they interact with ligands is crucial for understanding the fundamental biological processes in which proteins participate. It is also important to drug discovery efforts, particularly those focused on the development of therapeutic agents with protein targets.Mass spectrometry (MS) has become an increasingly useful analytical tool for acquiring biophysical information about the conformational properties of proteins in solution. A common strategy that has emerged for acquiring such information is to use matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI) MS to read out the results of solutionphase reactions that introduce covalent modifications into proteins. Two general types of MS-based covalent labeling strategies have proven useful for probing the biophysical p...

Determination of thermodynamic and kinetic properties of biomolecules by mass spectrometry

“…This strategy has been successfully applied for the analysis of protein conformational dynamics [39,41], folding/unfolding intermediates [42,43 ], ligand binding [44], and aggregation [45][46][47]. It is also worth mentioning here that HDX-MS can be applied to high-throughput screening of protein-ligand interactions and determination of binding affinity [48]. Methods, such as SUPREX (stability of unpurified proteins from rates of H/D exchange) [49 ,50-54] and PLIMSTEX (protein-ligand interaction by mass spectrometry, titration, and H/D exchange) [55][56][57][58], utilize the sensitivity of HDX rates to perturbations in protein structure by denaturing agents and/or ligand binding.…”

“…In a typical SUPREX setting, a series of protein or proteinligand complex samples is incubated for a certain time in a deuterated buffer containing increasing amount of a chemical denaturant, for example, guanidinium hydrochloride. Hence, the extent of hydrogen exchange is analyzed as a function of denaturant concentration and yields the free energy of folding DG f and the ligandinduced stabilization of protein structure DDG f , although with some limitations [48,54,59]. Recently, a very similar method in which labile deuterium labeling is substituted with stable oxidative labeling of methionine residues has been proposed -SPROX (stability of proteins from rates of oxidation) [48,[60][61][62].…”

“…Hence, the extent of hydrogen exchange is analyzed as a function of denaturant concentration and yields the free energy of folding DG f and the ligandinduced stabilization of protein structure DDG f , although with some limitations [48,54,59]. Recently, a very similar method in which labile deuterium labeling is substituted with stable oxidative labeling of methionine residues has been proposed -SPROX (stability of proteins from rates of oxidation) [48,[60][61][62]. In PLIMSTEX, the protein is titrated with ligand under native conditions and subjected to HDX for a certain, optimized time [48,[55][56][57][58].…”

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

“…Covalent labeling coupled with MS [29] can provide structural information on IMPs. Protein exposure to a hydrophilic reactive probe induces covalent modifications in solvent-accessible regions, whereas buried segments are sterically protected from the labeling agent [30]. The location(s) and the extent of labeling can be determined by LC-MS/MS peptide mapping [31,32].…”

Probing Protein Surface with a Solvent Mimetic Carbene Coupled to Detection by Mass Spectrometry

“…In this regard, parameters such as the heat capacity changes (ΔCp) between conformational states [7][8][9][10], the cooperativity of a conformational transition as measured by the slope (m) of the curve [11], and the changes in the radius of gyration (ΔRg) upon protein folding [12] are thought to be correlated with changes in the accessibility of the whole polypeptide chain, but do not directly yield local information. In a different vein, the profile of chemical reactivity of a given functional group present on an amino acid side chain against a set of reagents of different nature provides a description of the environment around this group, thus making possible to infer local solvent accessibility [13,14 and references cited therein]. However, by definition, selective chemical modification is limited to a single or a few chemical functionalities, rarely avoiding side reactions and frequently inducing conformational changes.…”