Using mass spectrometry (MS) to obtain information about a higher order structure of protein requires that a protein's structural properties are encoded into the mass of that protein. Covalent labeling (CL) with reagents that can irreversibly modify solvent accessible amino acid side chains is an effective way to encode structural information into the mass of a protein, as this information can be read-out in a straightforward manner using standard MS-based proteomics techniques. The differential reactivity of proteins under two or more conditions can be used to distinguish protein topologies, conformations, and/or binding sites. CL-MS methods have been effectively used for the structural analysis of proteins and protein complexes, particularly for systems that are difficult to study by other more traditional biochemical techniques. This review provides an overview of the non-specific CL approaches that have been combined with MS with a particular emphasis on the reagents that are commonly used, including hydroxyl radicals, carbenes, and diethylpyrocarbonate. We describe the reagent and protein factors that affect the reactivity of amino acid side chains. We also include details about experimental design and workflow, data analysis, recent applications, and some future prospects of CL-MS methods.
Amyloid aggregates are associated with several debilitating diseases, and there are numerous efforts to develop small molecule treatments against these diseases. One challenge associated with these efforts is determining protein binding site information for potential therapeutics because amyloid‐forming proteins rapidly form oligomers and aggregates, making traditional protein structural analysis techniques challenging. Using β-2‐microglobulin (β2m) as a model amyloid‐forming protein along with two recently identified small molecule amyloid inhibitors (i.e. rifamycin SV and doxycycline), we demonstrate that covalent labeling and mass spectrometry (MS) can be used to map small‐molecule binding sites for a rapidly aggregating protein. Specifically, three different covalent labeling reagents, namely diethylpyrocarbonate, 2,3‐butanedione, and the reagent pair EDC/GEE, are used together to pinpoint the binding sites of rifamycin SV, doxycycline, and another molecule, suramin, which binds but does not inhibit Cu(II)‐induced β2m amyloid formation. The labeling results reveal binding sites that are consistent with the known effects of these molecules on β2m amyloid formation and are in general agreement with molecular docking results. We expect that this combined covalent labeling approach will be applicable to other protein/small molecule systems that are difficult to study by traditional means.
Hydrogen–deuterium
exchange (HDX) mass spectrometry (MS)
and covalent labeling (CL) MS are typically considered to be complementary
methods for protein structural analysis, because one probes the protein
backbone, while the other probes side chains. For protein–ligand
interactions, we demonstrate in this work that the two labeling techniques
can provide synergistic structural information about protein–ligand
binding when reagents like diethylpyrocarbonate (DEPC) are used for
CL because of the differences in the reaction rates of DEPC and HDX.
Using three model protein–ligand systems, we show that the
slower time scale for DEPC labeling makes it only sensitive to changes
in solvent accessibility and insensitive to changes in protein structural
fluctuations, whereas HDX is sensitive to changes in both solvent
accessibility and structural fluctuations. When used together, the
two methods more clearly reveal binding sites and ligand-induced changes
to structural fluctuations that are distant from the binding site,
which is more comprehensive information than either technique alone
can provide. We predict that these two methods will find widespread
usage together for more deeply understanding protein–ligand
interactions.
Molybdenum carbides have been expected to be one of the promising catalysts for the hydrogen evolution reaction (HER) due to their similar d-band electronic structures to the Pt-group metals. However, the weaker hydrogen-adsorption ability of MoC severely hinders its applications. Guided by density functional theory calculations, we put forward a strategy to design the novel MoC-based electrocatalyst with surface reconstruction through sulfur doping. The incorporation of minor sulfur not only greatly increases the number of active sites and intrinsic activity but also optimizes the electronic structure to improve the electron transfer efficiency. As a result, the as-prepared sulfur-substituted MoC tackles the limitation of the Volmer step and exhibits superior HER performance with a small Tafel slope of 48 mV dec −1 . Theoretical investigations demonstrate that the terminal sulfur plays a critical role in facilitating a close to zero hydrogen adsorption energy (ΔG H* ) and a lower hydrogen release barrier.
The
combination of covalent labeling (CL) and mass spectrometry (MS) has
emerged as a useful tool for studying protein structure due to its
good structural coverage, the ability to study proteins in mixtures,
and its high sensitivity. Diethylpyrocarbonate (DEPC) is an effective
CL reagent that can label N-termini and the side chains of several
nucleophilic residues, providing information for about 30% of the
residues in the average protein. For DEPC to provide accurate structural
information, the extent of labeling must be controlled to minimize
label-induced structural perturbations. In this work, we establish
a quantitative correlation between general protein structural factors
and DEPC reaction rates by measuring the reaction rate coefficients
for several model proteins. Using principal component and regression
analyses, we find that the solvent accessible surface areas of histidine
and lysine residues in proteins are the primary factors that determine
a protein’s reactivity toward DEPC, despite the fact that other
more abundant residues, such as tyrosine, threonine, and serine, are
also labeled by DEPC. From the statistical analysis, a model emerges
that can be used to predict the reactivity of a protein based on its
structure and sequence, allowing the optimal DEPC concentration to
be chosen for a given protein. The resulting model is supported by
cross-validation studies and by accurately predicting of the reactivity
of five test proteins. Overall, our model reveals interesting insight
into the reactivity of proteins with DEPC, and it will facilitate
identification of optimal DEPC labeling conditions for proteins.
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