Investigating Therapeutic Protein Structure with Diethylpyrocarbonate Labeling and Mass Spectrometry

Borotto, Nicholas; Zhou, Yanyan; Hollingsworth, Stephen R.; Hale, John E.; Graban, Eric M; Vaughan, Robert C.; Vachet, Richard W.

doi:10.1021/acs.analchem.5b03180

Cited by 38 publications

(66 citation statements)

References 60 publications

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“…The modification percentage of each amino acid was determined in the same manner as described previously 21-25 Briefly, the ion abundance of each peptide of interest (i.e. labeled or unlabeled) was determined from extracted ion chromatograms (e.g.…”

Section: Methodsmentioning

confidence: 99%

“…Our group has found that diethylpyrocarbonate (DEPC) is a valuable pseudo‐selective reagent for studying protein/protein interactions. 19-25 DEPC has some advantages over other non‐selective reagents, such as hydroxyl radicals, in that it requires no special equipment (e.g. laser or synchrotron), and it results in only a single reaction product, thereby simplifying MS analyses and improving detection sensitivity.…”

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confidence: 99%

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Using Covalent Labeling and Mass Spectrometry To Study Protein Binding Sites of Amyloid Inhibiting Molecules

et al. 2017

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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.

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

Using Covalent Labeling and Mass Spectrometry To Study Protein Binding Sites of Amyloid Inhibiting Molecules

et al. 2017

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“…For reagents like DEPC that are inherently less reactive than hydroxyl radicals and carbenes, the effect of primary or tertiary structure might be expected to be more pronounced since changes in the pK a values of a given residue lead to changes in its protonation state and thus nucleophilicity. The relatively poor correlation observed between SASA and the DEPC reactivity of Ser and Thr residues [58, 59, 73] might imply that the microenvironment around these amino acids, as formed by a protein’s tertiary structure, tunes their reactivity.…”

Section: Factors Affecting Covalent Labeling Reactivitymentioning

confidence: 99%

Covalent labeling-mass spectrometry with non-specific reagents for studying protein structure and interactions

Limpikirati

Liu

Vachet

2018

Methods

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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.

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“…As a complement to free-radical footprinting, a variety of chemical reagents that target amino acid residues in a site-specific manner (e.g., N -ethylmaleimide 27 and diethylpyrocarbonate 28-29 ) can also provide information on site-specific solvent accessibility but react with protein substrates more slowly than do free radicals. 30 In this work, we performed carboxyl group footprinting with glycine ethyl ester (GEE) to corroborate the findings from HDX and FPOP, taking advantage of the presence of the many Asp/Glu residues in the flexible loops of IL-6R.…”

Section: Introductionmentioning

confidence: 99%

Orthogonal Mass Spectrometry-Based Footprinting for Epitope Mapping and Structural Characterization: The IL-6 Receptor upon Binding of Protein Therapeutics

Chen

et al. 2017

Anal. Chem.

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Higher order structure (HOS) is a crucial determinant for the biological functions and quality attributes of protein therapeutics. Mass spectrometry (MS)-based protein footprinting approaches play an important role in elucidating the relationship between protein biophysical properties and structure. Here, we describe the use of a combined method including hydrogen-deuterium exchange (HDX), fast photochemical oxidation of proteins (FPOP) and site-specific carboxyl group footprinting to investigate the HOS of protein and protein complexes. The work focuses on implementing complementary solution-phase footprinting approaches that differ in time scale, specificity for protein residue side chains vs. backbone as well as selectivity for different residue types to map integratively the epitope of human interleukin-6 receptor (IL-6R) for two adnectins with distinct affinities (Kd, Adnectin1 ∼ 6.2 pM vs. Kd, Adenctin2 ∼ 46 nM), and evaluate the resultant conformation/dynamic change of IL-6R. The suggested epitope, which is conserved for adenctin1 and adenctin2 binding, is a flexible loop that connects two β-strands in the cytokine-binding domain (DII) of IL-6R. We also found that adnectin1, the more strongly binding ligand, induces structural perturbations on two unstructured loops that are distally located beyond the epitope. Those changes are either attenuated or not detected for the case of adnectin2 binding. In addition to providing credibility in epitope determination, utilization of those combined approaches reveals the structural effects that can differentiate protein therapeutics with similar apparent biophysical properties.

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Investigating Therapeutic Protein Structure with Diethylpyrocarbonate Labeling and Mass Spectrometry

Cited by 38 publications

References 60 publications

Using Covalent Labeling and Mass Spectrometry To Study Protein Binding Sites of Amyloid Inhibiting Molecules

Using Covalent Labeling and Mass Spectrometry To Study Protein Binding Sites of Amyloid Inhibiting Molecules

Covalent labeling-mass spectrometry with non-specific reagents for studying protein structure and interactions

Orthogonal Mass Spectrometry-Based Footprinting for Epitope Mapping and Structural Characterization: The IL-6 Receptor upon Binding of Protein Therapeutics

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