Mapping the Energetic Epitope of an Antibody/Interleukin-23 Interaction with Hydrogen/Deuterium Exchange, Fast Photochemical Oxidation of Proteins Mass Spectrometry, and Alanine Shave Mutagenesis

Li, Jing; Wei, Hui; Krystek, Stanley R.; Bond, Derek; Brender, Ty; Cohen, Daniel N.; Feiner, Jena; Hamacher, Nels; Harshman, Johanna; Huang, Richard Y.‐C.; Julien, Susan; Lin, Zheng; Moore, K. T.; Mueller, Luciano; Noriega, Claire; Sejwal, Preeti; Sheppard, Paul W.; Stevens, Brenda; Chen, Guodong; Tymiak, Adrienne A.; Gross, Michael L.; Schneeweis, Lumelle A.

doi:10.1021/acs.analchem.6b03058

Cited by 73 publications

(78 citation statements)

References 59 publications

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“…We also showed that FPOP reveals fast fluctuations occurring remotely upon ligand binding, which is undetectable by slower footprinting methods 24 . Thus, we applied FPOP as a probe with high sensitivity to monitor changes in structure and dynamics of IL-6R.…”

Section: Resultsmentioning

confidence: 64%

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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Section: Resultsmentioning

confidence: 64%

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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“…Footprinting results show an overlap of important epitope regions detected by HDX-MS and FPOP. These results demonstrate FPOP and HDX-MS are equally useful for epitope structural mapping (78). In additional studies, FPOP, HDX-MS, and GEE labeling were used in tandem to demonstrate that the critical binding epitope of the IL-6/IL-R complex is the short segment 135 QNSPAED 141 .…”

Section: Comparison Of Protein Footprinting Methods Used To Study Higmentioning

confidence: 87%

“…For example, by using HDX-MS, FPOP, alanine shave mutagenesis (i.e. mutating potential key residues into alanine to study their effect on protein structure and functionality), and binding analytics in tandem, Li et al (78) reported the identification of an energetic epitope by determining the interfacial hot spot that dominates the binding affinity for an anti-interleukin-23 (anti-IL-23) antibody (Fig. 3).…”

Section: Comparison Of Protein Footprinting Methods Used To Study Higmentioning

confidence: 99%

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

Johnson

Stefano

Jones

2019

Journal of Biological Chemistry

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Edited by Wolfgang Peti Fast photochemical oxidation of proteins (FPOP) is a MSbased method that has proved useful in studies of protein structures, interactions, conformations, and protein folding. The success of this method relies on the irreversible labeling of solvent-exposed amino acid side chains by hydroxyl radicals. FPOP generates these radicals through laser-induced photolysis of hydrogen peroxide. The data obtained provide residue-level resolution of protein structures and interactions on the microsecond timescale, enabling investigations of fast processes such as protein folding and weak protein-protein interactions. An extensive comparison between FPOP and other footprinting techniques gives insight on their complementarity as well as the robustness of FPOP to provide unique structural information once unattainable. The versatility of this method is evidenced by both the heterogeneity of samples that can be analyzed by FPOP and the myriad of applications for which the method has been successfully used: from proteins of varying size to intact cells. This review discusses the wide applications of this technique and highlights its high potential. Applications including, but not limited to, protein folding, membrane proteins, structure elucidation, and epitope mapping are showcased. Furthermore, the use of FPOP has been extended to probing proteins in cells and in vivo. These promising developments are also presented herein.

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“…2) (55)(56)(57)(58). For the labeling studies, PB10 was at 2-fold molar excess relative to RiVax to favor complex formation.…”

Section: Hx-ms Analysis Of Rivax Bound To Cluster I Mabs (Pb10 R70 mentioning

confidence: 99%

High-Definition Mapping of Four Spatially Distinct Neutralizing Epitope Clusters on RiVax, a Candidate Ricin Toxin Subunit Vaccine

Toth

Angalakurthi

Slyke

et al. 2017

Clin Vaccine Immunol

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RiVax is a promising recombinant ricin toxin A subunit (RTA) vaccine antigen that has been shown to be safe and immunogenic in humans and effective at protecting rhesus macaques against lethal-dose aerosolized toxin exposure. We previously used a panel of RTA-specific monoclonal antibodies (MAbs) to demonstrate, by competition enzyme-linked immunosorbent assay (ELISA), that RiVax elicits similar serum antibody profiles in humans and macaques. However, the MAb binding sites on RiVax have yet to be defined. In this study, we employed hydrogen exchange-mass spectrometry (HX-MS) to localize the epitopes on RiVax recognized by nine toxin-neutralizing MAbs and one nonneutralizing MAb. Based on strong protection from hydrogen exchange, the nine MAbs grouped into four spatially distinct epitope clusters (namely, clusters I to IV). Cluster I MAbs protected RiVax's ␣-helix B (residues 94 to 107), a protruding immunodominant secondary structure element known to be a target of potent toxin-neutralizing antibodies. Cluster II consisted of two subclusters located on the "back side" (relative to the active site pocket) of RiVax. One subcluster involved ␣-helix A (residues 14 to 24) and ␣-helices F-G (residues 184 to 207); the other encompassed ␤-strand d (residues 62 to 69) and parts of ␣-helices D-E (154 to 164) and the intervening loop. Cluster III involved ␣-helices C and G on the front side of RiVax, while cluster IV formed a sash from the front to back of RiVax, spanning strands b, c, and d (residues 35 to 59). Having a highresolution B cell epitope map of RiVax will enable the development and optimization of competitive serum profiling assays to examine vaccine-induced antibody responses across species.KEYWORDS antibody, biodefense, epitope, mass spectrometry, toxin, vaccine R icin is one of a small group of plant and bacterial toxins that are classified at the domestic and international levels as potential agents of bioterrorism (1, 2). Ricin is a product of castor beans (Ricinus communis), which are cultivated globally for their oils that are used in industrial lubricants, cosmetics, and biofuels. The toxin itself is an ϳ65-kDa glycoprotein that makes up ϳ5% of the dry weight of a castor bean. The toxin can be purified by relatively simple affinity chromatography (3, 4). On a cellular level, ricin exerts its cytotoxic effects through ribosome inactivation and triggering of programmed cell death (5). Ricin's binding subunit, ricin toxin B subunit (RTB), is a galactose/N-acetyl galactosamine (Gal/GalNAc)-specific lectin that promotes toxin entry into mammalian cells, while ricin's enzymatic subunit, RTA, is an RNA N-glycosidase (EC 3.2.2.22) that, when successfully delivered into the cytoplasm, cleaves the sarcin-ricin

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Mapping the Energetic Epitope of an Antibody/Interleukin-23 Interaction with Hydrogen/Deuterium Exchange, Fast Photochemical Oxidation of Proteins Mass Spectrometry, and Alanine Shave Mutagenesis

Cited by 73 publications

References 59 publications

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

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

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

High-Definition Mapping of Four Spatially Distinct Neutralizing Epitope Clusters on RiVax, a Candidate Ricin Toxin Subunit Vaccine

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