Characterizing monoclonal antibody structure by carbodiimide/GEE footprinting

Abstract

Amino acid-specific covalent labeling is well suited to probe protein structure and macromolecular interactions, especially for macromolecules and their complexes that are difficult to examine by alternative means, due to size, complexity, or instability. Here we present a detailed account of carbodiimide-based covalent labeling (with GEE tagging) applied to a glycosylated monoclonal antibody therapeutic, which represents an important class of biologic drugs. Characterization of such proteins and their antigen complexes is essential to development of new biologic-based medicines. In this study, the experiments were optimized to preserve the structural integrity of the protein, and experimental conditions were varied and replicated to establish the reproducibility and precision of the technique. Homology-based models were generated and used to compare the solvent accessibility of the labeled residues, which include D, E, and the C-terminus, against the experimental surface accessibility data in order to understand the accuracy of the approach in providing an unbiased assessment of structure. Data from the protein were also compared to reactivity measures of several model peptides to explain sequence or structure-based variations in reactivity. The results highlight several advantages of this approach. These include: the ease of use at the bench top, the linearity of the dose response plots at high levels of labeling (indicating that the label does not significantly perturb the structure of the protein), the high reproducibility of replicate experiments (<2 % variation in modification extent), the similar reactivity of the 3 target probe residues (as suggested by analysis of model peptides), and the overall positive and significant correlation of reactivity and solvent accessible surface area (the latter values predicted by the homology modeling). Attenuation of reactivity, in otherwise solvent accessible probes, is documented as arising from the effects of positive charge or bond formation between adjacent amine and carboxyl groups, the latter accompanied by observed water loss. The results are also compared with data from hydroxyl radical-mediated oxidative footprinting on the same protein, showing that complementary information is gained from the 2 approaches, although the number of target residues in carbodiimide/GEE labeling is fewer. Overall, this approach is an accurate and precise method for assessing protein structure of biologic drugs. IntroductionMethods to describe structures of proteins and their higher order complexes continue to evolve and various advances have increased speed, efficiency, and resolution of structure determination.1 Most proteins behave according to the functiondictated-by-structure principle, and their proper conformation is central to their role in processes such as enzyme catalysis, cell signaling, or ligand binding. [2][3][4] Protein drugs (biologics) are one of the most important and fastest growing classes of drugs in the commercial marketplace today. The function and efficacy...

Mass Spectrometry-Based Protein Footprinting for Higher-Order Structure Analysis: Fundamentals and Applications

“…During the past decade, carboxyl group footprinting by GEE has been adopted in characterizing the structure of antibodies as well as their interaction with antigens and drugs. − In an informative example, Li et al applied orthogonal MS-based footprinting approaches to characterize the interaction between the extracellular region of human interleukin-6 receptor α-chain (IL-6R) and two types of adnectin (adnectin 1 and 2) that bind with picomolar and nanomolar affinity, respectively. Different from labeling in many other studies, GEE footprinting was executed in a time-dependent manner (Figure a–c) as commonly seen in HDX studies.…”

Rapid Online Oxidation of Proteins and Peptides via Electrospray-Accelerated Ozonation

“…An approach developed to overcome this limitation leverages solution-phase labeling of protein residues to encode the solvent accessibility and higher order structure of the protein into its mass. − MS is then utilized to assign the location of the modification. Due to their low sample requirements, rapid implementation, and applicability to proteins in membranes, disordered states, and complexes, these chemical labeling techniques have found a niche alongside the modern high-resolution structural techniques and have been widely utilized to address a diverse set of protein systems. − …”

Rapid Online Oxidation of Proteins and Peptides via Electrospray-Accelerated Ozonation

“…Due to their low sample requirements, rapid implementation, and applicability to proteins in membranes, disordered states, and complexes, these chemical labeling techniques have found a niche alongside the modern high-resolution structural techniques and have been widely utilized to address a diverse set of protein systems. [8][9][10][11][12][13][14][15][16][17][18][19] The reagents utilized in these workflows and hence amino acid selectivity, reaction rates, and necessary considerations can vary widely. 1,2,[14][15][16][20][21][22][3][4][5][6][7][8]12,13 A concern of many labeling-based technologies is the influence derivatization has on the conformation of the protein.…”

“…[8][9][10][11][12][13][14][15][16][17][18][19] The reagents utilized in these workflows and hence amino acid selectivity, reaction rates, and necessary considerations can vary widely. 1,2,[14][15][16][20][21][22][3][4][5][6][7][8]12,13 A concern of many labeling-based technologies is the influence derivatization has on the conformation of the protein. This is especially pertinent with these technologies, as many implementations generate multiply derivatized species and utilize reaction durations in the second to minute timescales.…”