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Keywords = apparent gas phase dissociation constants

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19 pages, 4541 KB  
Article
Mass Spectrometric ITEM-ONE and ITEM-TWO Analyses Confirm and Refine an Assembled Epitope of an Anti-Pertuzumab Affimer
by Claudia Röwer, Oladapo O. Olaleye, Rainer Bischoff and Michael O. Glocker
Biomolecules 2024, 14(1), 24; https://doi.org/10.3390/biom14010024 - 24 Dec 2023
Cited by 2 | Viewed by 2136
Abstract
Intact Transition Epitope Mapping—One-step Non-covalent force Exploitation (ITEM-ONE) analysis reveals an assembled epitope on the surface of Pertuzumab, which is recognized by the anti-Pertuzumab affimer 00557_709097. It encompasses amino acid residues NSGGSIYNQRFKGR, which are part of CDR2, as well as residues FTLSVDR, which [...] Read more.
Intact Transition Epitope Mapping—One-step Non-covalent force Exploitation (ITEM-ONE) analysis reveals an assembled epitope on the surface of Pertuzumab, which is recognized by the anti-Pertuzumab affimer 00557_709097. It encompasses amino acid residues NSGGSIYNQRFKGR, which are part of CDR2, as well as residues FTLSVDR, which are located on the variable region of Pertuzumab’s heavy chain and together form a surface area of 1381.46 Å2. Despite not being part of Pertuzumab’s CDR2, the partial sequence FTLSVDR marks a unique proteotypic Pertuzumab peptide. Binding between intact Pertuzumab and the anti-Pertuzumab affimer was further investigated using the Intact Transition Epitope Mapping—Thermodynamic Weak-force Order (ITEM-TWO) approach. Quantitative analysis of the complex dissociation reaction in the gas phase afforded a quasi-equilibrium constant (KD m0g#) of 3.07 × 10−12. The experimentally determined apparent enthalpy (ΔHm0g#) and apparent free energy (ΔGm0g#) of the complex dissociation reaction indicate that the opposite reaction—complex formation—is spontaneous at room temperature. Due to strong binding to Pertuzumab and because of recognizing Pertuzumab’s unique partial amino acid sequences, the anti-Pertuzumab affimer 00557_709097 is considered excellently suitable for implementation in Pertuzumab quantitation assays as well as for the accurate therapeutic drug monitoring of Pertuzumab in biological fluids. Full article
(This article belongs to the Section Biomacromolecules: Proteins, Nucleic Acids and Carbohydrates)
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16 pages, 3650 KB  
Article
Mass Spectrometric Analysis of Antibody—Epitope Peptide Complex Dissociation: Theoretical Concept and Practical Procedure of Binding Strength Characterization
by Bright D. Danquah, Kwabena F. M. Opuni, Claudia Roewer, Cornelia Koy and Michael O. Glocker
Molecules 2020, 25(20), 4776; https://doi.org/10.3390/molecules25204776 - 17 Oct 2020
Cited by 13 | Viewed by 3659
Abstract
Electrospray mass spectrometry is applied to determine apparent binding energies and quasi equilibrium dissociation constants of immune complex dissociation reactions in the gas phase. Myoglobin, a natural protein-ligand complex, has been used to develop the procedure which starts from determining mean charge states [...] Read more.
Electrospray mass spectrometry is applied to determine apparent binding energies and quasi equilibrium dissociation constants of immune complex dissociation reactions in the gas phase. Myoglobin, a natural protein-ligand complex, has been used to develop the procedure which starts from determining mean charge states and normalized and averaged ion intensities. The apparent dissociation constant KD m0g#= 3.60 × 10−12 for the gas phase heme dissociation process was calculated from the mass spectrometry data and by subsequent extrapolation to room temperature to mimic collision conditions for neutral and resting myoglobin. Similarly, for RNAse S dissociation at room temperature a KD m0g#= 4.03 × 10−12 was determined. The protocol was tested with two immune complexes consisting of epitope peptides and monoclonal antibodies. For the epitope peptide dissociation reaction of the FLAG peptide from the antiFLAG antibody complex an apparent gas phase dissociation constant KD m0g#= 4.04 × 10−12 was calculated. Likewise, an apparent KD m0g#= 4.58 × 10−12 was calculated for the troponin I epitope peptide—antiTroponin I antibody immune complex dissociation. Electrospray mass spectrometry is a rapid method, which requires small sample amounts for either identification of protein-bound ligands or for determination of the apparent gas phase protein-ligand complex binding strengths. Full article
(This article belongs to the Special Issue The Conformational Universe of Proteins and Peptides: Tales of Order and Disorder)
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